0.

0 LECTURE NOTES SECTION DAY OF COVERAGE

MOLECULAR TECHNIQUES
LECTURE NOTES 2013

BY DR. DAVID NG

ADVANCED MOLECULAR BIOLOGY LABORATORY

MICHAEL SMITH LABORATORIES
UNIVERSITY OF BRITISH COLUMBIA

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0.1 CONTENTS:

BY SECTION BY LECTURE DAY

1.1 DNA: General Info

1.2 DNA: General Workflow Before the start of workshop, please read
APPENDIX A (on replication)
2.1 DNA: Genomic/Plasmid
2.2 DNA: Cell Lysis
2.3A DNA: Nucleic Acid Purification MONDAY
2.3B DNA: Nucleic Acid Purification 1.1, 1.2
2.4 DNA: Precipitation 8.1, 8.2, 8.3, APPENDIX B
2.5 DNA: Quantitation 4.1A*, 4.1B*, 2.6*
2.6 DNA: Agarose Gel Electrophoresis
TUESDAY
3.1 CLONING: What is a Vector? 4.1A*, 4.1B*, 2.6*
3.2 CLONING: Choosing a Vector I 2.1, 2.2, 2.3B, 2.5
3.3 CLONING: Choosing a Vector II 3.1, 3.2, 3.3
3.4 CLONING: Plasmid Preps 4.2, 5.1, 6.4*

4.1A ENZYMES: RE Digests I WEDNESDAY

4.1B ENZYMES: RE Digests II 7.1, 7.2, 7.3
4.2 ENZYMES: Ligases/Phosphatases 2.3A, 2.4
6.1, 6.2
5.1 TRANSFORMATION
THURSDAY
6.1 PROTEINS: General Info 3.4
6.2 PROTEINS: SDS PAGE 2.3B
6.3 PROTEINS: Western Blot 6.4
6.4 PROTEINS: 2D Gel Electrophoresis 8.4

7.1 RNA: General Info

7.2 RNA: Isolation and Purification FRIDAY
7.3 RNA: Gene Expression 6.3

8.1 PCR: The Basics

8.2 PCR: The Specifics
8.3 PCR: Troubleshooting
8.4 REAL TIME PCR

APPENDIX A: Replication
APPENDIX B: Hybridation/Stringency
1.1 DNA: General Info. DAY 1
First off, some basic things to consider that ultimately affect how you envision the behaviour of
DNA in your experiments:

1. DNA is a simple molecule No really! it’s got your A, T, C and G, which from the eyes of
a chemist, are pretty similar anyway. What does this mean? It means “predictability”
which is a very handy thing in research.
2. DNA is negatively charged Very much so – lots of electrons whizzing around those
oxygen groups.
3. DNA has lots of ring structures You remember things like pi orbitals, and the difference
between polar and non-polar bonds? Anyway, the point is that ring structures are
generally quite hydrophobic.
4. DNA is a very robust molecule. Spat in your sample? Probably o.k. Left out at room
temperature over the weekend? Not a problem. Generally, the easiest of the biological
macromolecules to work with).
5. DNA work has lots of toys. (Talk about accessories! You simply have a lot of options
as far as what you can do with this molecule, whether it is a procedure, or a specific
enzyme, etc)

Some general common sense rules that are worth applying to all DNA experiments, and frankly,
while we’re at it, to all molecular experiments.

1. Know the experiment’s level of forgiveness. Another way of saying that it pays to
know the chemistry of your procedure. Inevitably, each experiment has a degree of
forgiveness, which is a really useful thing to know. This allows you to gauge your level of
care, which in turn will reflect on your efficiency as well as your ability to troubleshoot.
2. Get your sample as pure as you can. Although this is related to point one, consider the
following thought: any impurity is an outright invitation for caveats. i) If you have
contamination, your subsequent steps may be hindered, or worse, unduly affected. ii) If
you have a contamination, you might just lose your sample outright. i.e. DNA doesn’t like
nucleases. And nucleases are “everywhere.”
3. If you had to choose, be gentle rather that be rough. Doesn’t hurt to be careful when
handling material. i.e. keep everything cold, since these enzymes are much more active
at physiological temperatures. (i.e. use of "ice cold" this and that") Wear gloves, etc.
4. Know the idiosyncrasies of your molecule: At times, you need to be aware of specific
nuances that apply to your particular “brand” of molecule. For instances, genomic DNA is
different from plasmid DNA is different from a PCR product is different from an EST
fragment.
5. Think carefully about how much stuff you actually need. Small amounts generally
easier to work with, machines are smaller, take less time to operate, etc, etc, etc… So, if
you know, you don’t need much, then only use that amount.
1.2 DNA: General Workflow. DAY 1
In general, when doing molecular work with DNA sequences, there are a number of basic things
you’re trying to do. Often, instructional manuals will attempt to classify this ilst as a “general
workflow” which is akin to a sort of flowchart of events.

In a nutshell, this tends to involve the following:

1. Figure out what it is you’re interested in: This step tends to be based on literature search, or
via an observation that one has deemed intriguing. Basically, this narrows the boundaries of the
DNA code you’re keen to explore. A defining feature of this step is also figuring out where this
sequence might come from, as well as its likely size. Both of these parameters will greatly
influence subsequent steps.

2. Physically retrieve the sequence that you are interested in: Once you know what you’re
interested in, next comes the actual work to get it. This might involve working with tissue/cells
which tends to incorporate various cell lysis and nucleic acid purification procedures. Or it may
entail working with material that is provided in purified form as a pre-determine lab sample. As
well, there are often steps to modify this DNA sample so that it can be used for subsequent
steps,: this might include finessing its size, or simply amplifying it to appropriate amounts. Note
that the most common way of performing all of these steps often involves the Polymerase Chain
Reaction method.

3. Insert your sequence of interest into an appropriate vector. Although this will be described
more fully, harbouring your DNA of interest in a specific vector will define how you intend to
“characterize” your sequence of interest. i.e. Do you want to sequence it? Do you want to
transcribe/translate into a protein? Do you want to insert your sequence into a host cell for
observation? etc.

In all, this workshop will cover a wide variety of different techniques that provides a myriad of
different options for you to cover this “general workflow.”

Example Workflow

> GET TISSUE

> LYSE CELLS
> PURIFY GENOMIC DNA
> PRECIPITATE TO CONCENTRATE
> PCR DESIRED FRAGMENT
> CHECK ON AGAROSE GEL
> PURIFY FRAGMENT / QUANTITATE
> LIGATE INTO VECTOR
> SEQUENCE VECTOR (TO CHECK FRAGMENT)
> TRANSFORM/TRANSFECT INTO HOST
2.1 DNA: Genomic/Plasmid DAY 2
We’ll talk later about plasmids, so will focus primarily on genomic DNA.

Genomic DNA? What is it? Well, it’s actually trying to retrieve “all” of the sequence found within
an organism. Wiki also says it succinctly:

“In modern molecular biology and genetics, the genome is the entirety of an
organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA.
The genome includes both the genes and the non-coding sequences of the DNA/RNA.”

From a practical point of view, if you’re working with bacterial or eukaryotic samples, you are
trying to isolate all DNA from your sample. By the way, many species of genomic DNA samples
involve big, complicated pieces that often end up looking like snot in solution.

Why are you getting genomic DNA? (a couple of reasons)

1. Profiling/Fingerprint: This is the stuff you use to compare blood/semen samples. Essentially,
your genomic DNA can lead to some form of reproducible data that provides a unique
representation of the sample (i.e. band profile on a PCR DNA fingerprint for example)

2. First attempt at cloning a new gene. If you plan on looking for something completely new
and novel. Therefore, you will need to go to the original source of that code which is the genome.
And although, you may be able to get protein or mRNA information before this step, you will
ultimately need to define it within a genomic context (i.e. what chromosome is it on, promoter
regions, intron/exon organization, etc etc.)

3. Characterization/Diagnotics. Lots of examples in basic research. I.e. you’ve made a

transgenic/knockout organism, and you need to check if indeed, the modification has occurred –
this starts with a genomic prep. You have an interesting phenotype and you want to look deeper.
i.e. a disease state. This hunt may often start from a genomic prep.

4. Studying genome for clues into gene expression and regulation: Although, usually the
cDNA for a gene gets a lot of attention (this is a DNA copy of the messenger RNA directly
responsible for coding the protein), this edited form of the gene misses out a lot of stuff that may
be interesting or relevant to your research. I.E. Study gene expression regulation, euchromatin
nuances, tissue/developmental specificity, spliced isoforms, etc.....
2.2 DNA: Cell Lysis. DAY 2
IT'S IMPORTANT TO REALIZE that there are many variations of the lysis procedure. Some are
quicker, some are more efficient, some are more expensive, some only work in certain situations.
In our PCR experiment, we performed a simple boiling step to get the job done.

If there’s time in class, we’ll also mention the SDS/proteinase K method. It being a very standard
procedure (often embedded in kits).

NOTE: Large pieces of Genomic DNA can shear. (not actually a big deal for most procedures
but why take the risk). In this case, dealing with a piece of DNA that is ~100kb + in size. No
vortexing, no vigorous pipetting/snip the end of your tip before use.

EXAMPLE OF COMMON LYSIS PROCEDURE: (SDS/Proteinase K):

1. Place the cells in a “physiological” like buffer. There are many varants of this, but the
below is a good example.
Tris (buffering agent -> good @pH6-8) Often at the 10 – 20mM range.
EDTA chelates divalent cations which are necessary cofactors for DNase activity (way of
shutting down nucleases) Usually at the <10mM range.
NaCl at physiological concentration (generally considered to be 100 – 150mM) Keeps all
molecules happy (particularily proteinase k)/prevents unwanted aggregation.

NOTE that TE buffer is a commonly used buffer which contains the aforementioned Tris
and EDTA.

2. Then add the reagents which are responsible for the lysis.

SDS, nasty ionic detergent/ good at breaking membrane, general denaturant (inhibit
enzyme activity). Since DNA is so robust, not really adversely affected by SDS treatment.
Note: When dealing with plant material, a very common detergent in place of SDS is
Sarkosyl. This essentially behaves in a similar manner to SDS.
Proteinase K->serine protease (works well at 55C), used because it is very effective and
not particularily susceptible to SDS, and other denaturants such as urea. Proteinase K
will chew up protein, which helps lysis in general and frees up the DNA from any protein
gunk associated with it (euchromatin structures/histones, etc). Best used FRESH (*quite
an important step).

Incubation step generally a minimum of an hour. Although most will allow the procedure
to go longer (this may be largely dependant on material being used – i.e. many
procedures outline an overnight or >16hrs incubation time). I.E. mouse tails -> may want
to go overnight).
2.3A DNA: Nucleic Acid Purification. DAY 3
There are a number of different ways to purify nucleic acids (indeed, with our PCR
experiment we are just using special CHELEX beads which are designed to pull out
positively charged ions). For the remainder of the course, we’ll be utilizing three other
methodologies described below.

A. DIY METHODS

PHENOL/CHLOROFORM

NOTE that ***Phenol*** is pretty nasty! It will burn. (Apparently, someone once told me that if you
cover 5% of your body, you will die). Since we are using reasonable amounts, do PHENOL
addition in fumehood, but all manipulations can occur at the nech. If you get it on you -> not panic
mode. Quickly rinse off with cold water.

Phenol, by the way, is usually buffer saturated. (i.e. when you buy it or make it, the solution
comes in two layers -> top layer is excess buffer, and bottom layer is buffer saturated phenol.
This is because the pH is important - In order for our purification to work, we need the PHENOL
at a neutral pH.( acidic pH is not good as DNA becomes soluble in phenol). The fact that it is
saturated is also important because this means that any additional aqueous (i.e. water) solution
you add will create it’s own fluid layer.
This purification procedure works on the principle of "differential solubility".

1. To your lysate, you will add an equivalent volume Phenol/Chloroform/Isoamyl alcohol.

(usually at a volume ratio of 24:23:1). Phenol – organic solvent/ nucleic acids not
soluble at all. Therefore, DNA/RNA will stay dissolved in aq phase. Lipids and
polysaccharides preferentially go into the phenol phase. Proteins will also selectively go
into phenol solution.
2. FURTHERMORE phenol also acts as a denaturant, proteins denature form aggregates
and will collect at the interface. You will see GUNK @ the interface.
3. Choloroform, also has same general attributes as phenol (as far as solvent properties)
but also stabilizes the rather unstable boundary between aq and organic layers. Isoamyl
alcohol also contributes to interface stability and also helps prevent frothing.

Generally you do this step ~2 or 3 times. The more times you do it, the cleaner your sample (you
may even note that the interphase gets cleaner and cleaner with each step). Note this procedure
is very reliable and does not lose much DNA yield. This is probably why a lot of labs still like to
use it. (BACK EXTRACT: adding extra aqueous to your organic samples).

Sometimes do a final Choloform step. Here, the interface is a little trickier to handle. But this step
is a good safeguard to prevent any organic carrying over to your final aqueous solution. Likely
finish with a chloroform step because it evaporates easily (?). In other words, this give you the
option of leaving the lid open to really make sure ALL your organics are gone from your prep.
2.3B DNA: Nucleic Acid Purification. DAY 2/DAY 4

B. KIT BASED METHODS

pI BASED KITS

We’ll first be checking out Invitrogen’s ChargeSwitch kits, which rely on magnetic beads that
contain polymers with neutral pI. Basically, if the environment is such that the net charge of the
beads can bind to the DNA, you can release the DNA by altering the pH environment of the
solution in the other direction (i.e. change the charge of the polymer). In short:

If pH < pI (more acidic), then the net charge of polymer linker becomes more positive
(higher affinity for DNA)

If pH > pI (more basic), then the net charge of polymer linker becomes more negative
(lower affinity for DNA)

Fast! But you would need to invest in special magnet racks.

SILICA BEAD BASED KITS

(i.e. QIAprep) Nucleic acid purification kits more commonly involves the use of silica based
beads which are specifically designed to interact with the very electrostatically charged nucleic
acid molecules.
Our QIAGEN preps are a good example of how kits work in general:

1. Our DNA sample was first treated with a salt solution (something like NaI). This stuff is
usually classed as a chaotropic salt, which is really a fancy way of calling a salt that is
capable of altering structures by interacting with and thereby sequestering many
molecules of water. In other words, salts like NaI can bind to several molecules of water
at high stoichiometry, depriving water from the DNA structure in particular. This will
alter its shape/charge/etc which makes it specifically bind to the silica beads (often
pretreated themselves with ions to make them more amenable to specific binding).
2. We next do a wash, which in our case is called NEW WASH buffer. Not entirely sure
what this is, but you can bet it doesn't affect the electrostatic state of your nucleic acid
(likely an alcohol/water mixture). You don't want 100% water during the wash steps, or
else your DNA will get the water back and fall off the beads!
3. Which is why the final elution steps are water alone.

THERE are lots of variations of these kits. Popular these days are versions that are set up in a
column format. Arguably, most popular are those sold by Qiagen.
2.4 DNA: Precipitation. DAY 3
PRECIPITATION OF DNA (alcohol and salt procedure)

To precipitate nucelic acids, you can add a high concentration of salt (i.e. Sodium Chloride or
Ammonium Acetate). Why? Helps in the precipitation of the DNA in EtOH. NOTE: sometimes,
you can also omit salt entirely (dependant on concentration of DNA). Salt will help neutralize
negative charge of DNA (will also sequester the solvent molecules - in this case water) Salt will
also interact with water, thereby weakening it’s solvating prowess. This is commonly known as
“salting out”

Use 100% EtOH. Time frame (show graph) EtOH generally helps because it is a much crappier
solvent than something like water (which is very polar). DNA will tend to stay precipitated in
>65% Ethanol. NOTE that efficiency of EtOH precipitation is dependant on a number of things.
Temp, time, amount of DNA.

Can also use isopropanol for precipitation steps. RNA tends to stay soluble in this solvent
(selective precipitation). which is why some people use it for this purpose. DNA will tend to stay
precipitated in >50% isopropanol.

Once precipitated, DNA will tend to stay insoluble if kept in >70% ethanol. Hence, a lot of
washing steps tend involve Ethanol based solutions, which is important as it removes any excess
salt present in your sample. Final resuspension of sample in TE.

Graph below highlights how, generally speaking, unless working with very small amounts, DNA
precipitates pretty quickly.
2.5 DNA: Quantitation DAY 2
Spectrophotometry readings: Using UV absorbance to ascertain DNA/RNA amounts and
purity. Ring structures can absorb UV wavelengths. BUT Lots of things have ring-structures
(including nucleic acids, proteins, organics, detergents, lipids, the list goes on and on...)

i.e. Both DNA and RNA -> ring structure absorbs strongly at ~ 260nm and ~280nm.
Protein => some amino acids like W and P and Y also flouresce strongly at ~ 260nm and
~280nm.

Bottom line is that you have to take numbers with a grain of salt. Very rough estimate, since lots
of things absorb at UV wavelengths. Also quite sensitive to pH which is why dilutions are often
done with TE buffer at a particular pH, that was also used to calibrate the spectrophotometer.

You can also assess purity by looking at ratios of 260 over 280.
DNA A260/A280 ~1.8
RNA A260/A280 ~2.0

These are good ratios for purified product. If you get good numbers here, then maybe you can
also get good quantitation numbers (NOTE: that the conversion for O.D. numbers to DNA
amounts is the constant 50ug/ml per O.D260nm value, LIKEWISE, the constant for RNA is
40ug/ml per O.D.260nm value.)

Other wavelengths of interest include: A230 (good especially when phenol is in the mix, since
phenol absorbs most strongly at 270nm).

Quantitation Assays: Usually rely on stoichiometry binding between reagents like Ethidium
Bromide, SYBR dyes to correlate with a DNA amount. Generally a more robust, more expensive
manner of determining amounts. ALONG the same lines, DNA fragment quantitation can be
done by comparing band intensities in an ethidium bromide stained gel – i.e. use a marker as a
gauge.
2.6 DNA: Agarose Gel Electrophoresis. DAY 1/2
Using Agarose. Polysaccharide polymer. Used because of its ability to form pore sizes capable
of resolving ~ 0.2kb to ~60kb, (200bp to 60,000bp).
Essentially, creates a big mesh of fibers that your DNA has to pass through. Electrical charge is
the driving force and things will separate according to size.

DNA works well in this set-up because for things to separate in correlative manner, all DNA
species generally have about the same charge density. ALSO, in our case, the DNA we are
looking at has been cut with restriction enzymes - therefore all DNA fragments are predominantly
linear in shape.

Loading buffer:
Glycerol: thickens sample up so that it doesn’t float away after you load it into the well
0.1 M EDTA stop reagent for the assay
1% SDS help denature the RE stop the reaction
0.1% bromophenol blue. Dye. Helps you visualize sample when loading. Will run towards
+electrode. Can use it as a rough idea of where your DNA may be running (dependant on gel%)

Running buffer:
Tris Borate EDTA (TBE) In this case, borate is your ion, which allows the generation of an
electric field in the gel set-up.
Your common alternative is TAE: Here, acetate acts as an alternative ion - is often used because
it works and is much cheaper. Need 50c for 10L of TAE, (need $10 for 10L of TBE)

But tris/borate has a significantly better buffering capacity, which means gel running is more
reliable especially at high voltages (for speed), or long running times (i.e. overnight). BUT
borate (when preparing the gel in microwave or oven) also forms complexes with the agarose
sugar monomers/polymers. Can be a problem if using procedures to isolate band from a gel (i.e.
melting of gel is required), although most band extraction kits come with chemistry to deal with
this.

Quick mention of Sodium Borate systems (cheaper, very fast, but resolution can be affected –
Kern and Brody, BioTechniques Feb 2004)

Visualizing the DNA:

Most people now use SYBR stains Examples include SYBR SAFE,/SYBR Green I/ SYBR
GOLD which are exceptionally sensitive nucleic acid gel stains with bright fluorescence when
bound to dsDNA and low background in gels, making it ideal for detecting dsDNA in gels using
laser scanners or standard UV transilluminators. Compared to Ethidium Bromide, these stains
are generally about a hundred fold more sensitive, and less carcinogenic to boot! (a bit more
expensive mind you, and you will need a particular filter in your light box to see it – although this
isn’t that expensive). Stain can occur during run, or after run.

Alternatively, you also have Ethidium Bromide (carcinogen), also sensitive stain that
interchelates DNA (which has the added ability of slightly uncoiling it). Need to use a UV lightbox
to see it – take care to use appropriate eye shielding.
Some labs add EtBr into gel. Some add it after gel has run (i.e. stain with solution containing
EtBr) -> adding it into the gel is much easier, but if the apparent molecular weight of closed
circular DNA is particularily important to you, it may be worth adding after so that it doesn’t affect
its molecular weight.
FOR EtBr, you should inquire at your health, safety department, as many research facilities are
phasing out EtBr use.
3.1 CLONING: What is a Vector? DAY 2
Vectors are essentially shuttles that carry your DNA. At the most basic level, almost all vectors
have the following traits (sorry for the long long titles):

UPKEEP: Naked DNA (no matter how important it is to your research) does not upkeep itself.
Therefore, to do this, you need your “DNA of interest” to replicate independently of integration
into host genome. i.e. the vector allows the use existing replication machinery of host organism.
OR another way to get around this, is for the vector to contain information that can drive
integration into the host genome.

PLACE TO PUT DNA IN: all vectors generally have defined places where you can insert your
DNA of interest. This is usually at a place called the Multiple Cloning Site (MCS), and is
governed by (i) convenience (i.e. easy to put something in this area), or (ii) geography (you put
your DNA “here” because it is next to some element – say a promoter – that you want to use to
act on your DNA of interest.” Vector nomenclature, while hardly poetic, is usually a reflection of
what the MCS and it’s surrounding geography is all about.

SOMETHING THAT LETS YOU KNOW IF THE VECTOR IS IN THE ORGANISM:

All vectors will generally contain a selectable marker (notable exception is where GM crops are
involved). This is usually a gene that confers resistance to some sort of drug. Commons ones
used in research field include antibiotics such as ampicillin (common for [Link]), and neomycin
(common in mammalian cell cultures). On the other hand, some organisms will rely on
auxotrophic traits. This turns up a lot in yeast work, whereby host cells are mutant and unable to
make a nutrient like tryptophan. Sooo... In order for these strains to grow, you either need to
supply it in media, or you insert a vector into the yeast that reconstitutes the ability to make trp..

SOMETHING THAT LETS YOU KNOW IF THE DNA OF INTEREST YOU PUT IN IS
ACTUALLY IN THE VECTOR, WHICH IN TURN IS IN THE ORGANISM:
There are a number of strategies that let you determine whether your DNA of interest actually
made it into the vector. These strategies often entail some form of insertional inactivation, or
ligation trick (i.e. CIP assay, T overhang systems).
3.2 CLONING: Choosing a vector I DAY 2
There are two general things to think about. First of all is the principle that size matters. i.e. How
big is my DNA of interest? Is it a relatively small <10,000bp cDNA, or is it a chunk of
chromosome >300,000bp? In this respect, here is a run down of common vector types:

1 PLASMID VECTORS: such as pUC18. (used in our experiment):

a) selectable marker: ampicillin resistance cassette. Therefore if you want to ensure that your
bug has gotten the plasmid, then you just test for the bug’s ability to survive under drug treatment.
b) Blue white screening: which sort of works like this -> Things to point out ->LacZ gene (b-
galactosidase) Bugs will constitutively express a lac repressor. Which interacts with lacUV
promoter. Therefore, no transcription of beta-galactosidase. But if you add lactose -> lac
repressors fall off promoter. What you usually use is IPTG (isopropyl-b-D-thiogalactoside)
which is an analog of lactose.
Anyways, if there is NO insert -> lacZ gene will get transcribed. Codes for a b-galactosidase.
If there is an insert -> something else will get made. Frameshift mutations/insertional inactivation.
Will not get lacZ transcribed.
c) MCS: Small area of the plasmid where the vector designers have decided to put lots of unique
restriction sites in a small area. I.e. try to make it as easy as possible for you to have a
convenient nice restriction site that works for your cloning strategy.

2 BACTERIOPHAGE VECTORS: (very old school – but will cover as background necessary):
fancy word for virus that infects bacteria. Good for moving around ~20kb worth of DNA. Good
example of bacteriophage vector is the "lambda DNA."
This thing is about 49kb in size, containing essential and non-essential regions as outlined in
figure. Essentially, the key idea is to take advantage of the viral pathways to get your DNA inside.
I.E. you can package your DNA into a bacteriophage structure, which can infect and shunt your
DNA into the host cell (a bug). ONCE the DNA is inside, the bacteriophage will still go about
making more DNA, and making all the necessary components to make more of itself. This is
essentially how you upkeep your DNA.
The only technical difficulty is how you would "harvest material" . i.e. your DNA is not in the
bacteria per se, but rather is inside the bacteriophage, and the logistics of getting bacteriophage
cultures are more difficult (i.e. plaques on a plate)... I.E. WHY phage systems are now rarely
used. The other reason is because of...

3. COSMID VECTORS: Good for packaging 30-45kb of material. Think of it as an unworthy

bacteriophage. I.e. Even essential regions have been deleted (this is why it can carry more than
normal bacteriophage vectors) but special cohesive sites (COS SITES) are still present.
Therefore, still has enough genetic information to get packaged but not enough to "be a
bacteriophage" i.e. doesn’t have information to make more bacteriophage.

NOW you can use a KIT to supply materials (bacteriophage bits and packing machinary) needed
for packaging of your DNA into a "pseudo" bacteriophage. Then your bacteriophage can infect a
host organism, and effectively transform it (deliver your DNA). ONCE, DNA gets in, the COS sites
will direct recircularization and you now essentially have a BIG PLASMID (therefore can use
normal plasmid techniques for isolation).

4. ARTIFICIAL CHROMOSOME SYSTEMS – BACs and YACs: "The realization that the
components of a eukaryotic chromosome that are required for stable replication and replication in
YEAST, are VERY SMALL very DEFINED sequences” CAN TAKE UP TO 1 Mb!!
NOTE that BACs or Bacterial Articifical Chromosomes (sometimes known as PACs or
plasmid artificial chromosome) are more common nowadays. Similar to YACs in principle in
that they allow incorporation of a huge amount of DNA (up to 300kb). The difference is that you
can work in e. coli which is much easier than yeast manipulation
3.3 CLONING: Choosing a vector II DAY 2
The other constraint, of course, is what you want to do with your DNA of interest, and quite
literally, to say that the world is your oyster is very much a reality. Here are two examples of the
sorts of things you need to think about.
3.4 CLONING: Plasmid Preps. DAY 4
Isolating plasmid DNA from other types of DNA (i.e. genomic) is actually very simple. In short, it
usually involves a denaturing step, followed by a quick renaturing step. The IDEA is that plasmid
DNA being much smaller, can renature relatively easily - consequently, once back to normal it
can go into solution easily. Something like genomic DNA will have an incredibly hard time
renaturing because it is simply too big and too complicated. It doesn't renature effectively and
instead tangles up and precipitates out.
IF YOU THINK ABOUT IT, you have now separated your plasmid (in solution) from your genomic
(out of solution) prep. You simply have to centrifuge away the genomic pellet, and you are left
with your plasmid DNA (+ all the other cellular crap like proteins, etc etc --> however, now you
can use any standard DNA purification procedure).

Most common way of doing this is known as the ALKALINE LYSIS METHOD (which also makes
an appearance in practically all kit based plasmid prep methodologies)

Here the idea is to chemically denature and renature.

1. Need to open the cells up. NaOH and SDS. Ruptures cells, and denatures everything. Low
pH specifically breaks H bonds in dsDNA.

O.K. so your test tube is now this messy mix of denatured stuff.
Genomic DNA (big) -> denatured.
Plasmid DNA (small) -> denatured.
Proteins -> denatured.

THROW IN salt that is acidic (KAc pH4.8).

Salt helps in the precipitation process. Acid -> causes things to go back to neutral. DNA can
renature BUT HAPPENS VERY QUICKLY.

Large DNA renatures as a MESS. Small DNA renatures O.K.

So, genomic DNA will precipitate out (should see a white mess), but your plasmid DNA will now
be in solution. TA DA!
Move onto purification/precipitation step.

ALTERNATIVE QUICK AND DIRTY PLASMID PREP METHOD:

Via causing the cells to lyse by using STET + lysozyme. and then the trick is to boil and then cool
(this provides the denature and renature step)
4.1A ENZYMES: RE digests I READ ON YOUR OWN
Generally speaking, molecular biology enzymes are like your high maintenance buddies. It really
pays to know them well, and they will end up being very useful to you. We will focus on a lot of
details with regards to Restriction Endonucleases (REs), so that you’re aware of the sorts of
nuances entailed.

NOTE: Enzymes are pretty expensive so you want to keep them cold and keep them clean. This
means you always use a freezer box when keeping them at your bench. This means that if you
don’t have a freezer box, you do everything at the freezer! This means no double dipping.
NOTE: bug a vendor and get some free catalogs – these are very useful reference source.
Especially useful is the New England Biolabs catalog.

1. WHAT IS A RE?: Think of it as an enzyme that cuts or degrades DNA but in a very specific
manner. There are actually several classes of REs, but the most commonly used are
affectionately categorized by the type of cut they leave. In shop talk, the two common types are
referred to as “sticky end” and “blunt end” cuts.
In terms of physiological or historical background:
*All started in 1970. Hamilton Smith at John Hopkins University studying Hemophilus influenza. ->
extracts from this bug could cleave DNA at very precise points. Turns out, bugs have sets of
restriction enzymes which are believed to serve as their "immune system" of sorts protecting
them from virus infection (viral DNA gets cleaved). To date well over 3000 diff REs to choose
from, which were found from screening >10000 bacteria.

2. WHY ARE YOU USING A RE?: Generally speaking, you are trying to create a specific cut,
often to obtain a particular fragment or chunk of DNA. However, RE can be used to simply “alter”
the size of a DNA sample to fulfill a particular purpose – a good example is to use a RE to cut
genomic DNA down to size for effective agarose gel viewing. In some respects, this ties into the
idea of WHICH ONE TO USE?

3. HOW MUCH TO USE?: Amount? Very simple. 1 unit of enzyme is enough (theorectically) to
do whatever it’s suppose to do (in this case “cut”) 1ug DNA in a specific amount of time at the
defined temperature (in a reaction volume <50µl)
BUT it’s interesting to point out that every enzyme can come with its own personality. (which, in
turn, can be dependant on the batch or company they come from). Common restriction enzymes
generally work quite well, and arguably, any enzyme that is not “fancy” is more reliable.
NOTE that generally speaking blunt cutters seem to be more finicky than sticky cutters.
ALSO NOTE that researchers often add more enzyme than is theorectically needed (likely to
ensure that the reaction will go to completion, but probably it’s simply impossible to pipette
anything less than 1.0µl accurately anyways).
4.1B ENZYMES: RE digests II READ ON YOUR OWN
4. CONDITIONS OF THE REACTIONS: enzymes will always come with "special" buffer.
Generally, the buffer is all about making your enzyme happy - keeps it at a particular pH (usually
around 7 to 8), a particular salt concentration, and has cofactors like Mg in it.

Salt is the most varied consideration in getting a reaction to work (In fact, in the not so recent
past, buffers use to be separated simply as low, med and high salt.). This is particularily true if
you wanted to cut your DNA with >2 different REs (double digest). In the past, you would cut first
with RE that worked at lower [salt] / bring up the [salt] for second enzyme. (this would be an
example of a tough dilution question)
Nowadays, there are handy dandy buffer activity charts – lists % activity for an enzyme under
different buffers.

5. OTHER THINGS YOU CAN ADD. RnaseI – get rid of extra RNA in your prep. BSA –
(albumen) generic protein. Proteins have an uncanny ability to destabilize when solvated in a
VERY dilute concentration (why? Too much water solvating protein) Companies try to fix this by
adding more (generic) protein in solution so that overall [protein] is higher. Spermidine (* not
used as much anymore): Added b/c of its ability to interact with polymerases and other DNA
binding proteins. DTT: reducing agent. Dithiothrietol -> during purification process by company ->
May get unwanted disulfide bond formation, which would affect enzyme structure.

6. TIME AND TEMPERATURE: only guidelines. In terms of RE, the unit value is usually under
time constraint of 1 hour. Therefore, 1 hour is the bare minimum. However, it is considered safe
to leave digests going overnight, which is often suggested if it is crucial for you to get complete
digest. Some enzymes sensitive to high temperatures: useful if you want to inactivate it (heat
inactivation).

7. QUALITY OF YOUR DNA SAMPLE : As a whole, restriction digests are very forgiving. BUT
no organics (i.e. phenol or chloroform), as they will affect RE viability.
METHYLATION: will also affect the ability to be cleaved. IN actual fact, this is what protects the
bacteria from its own restriction enzyme defence system. I.e. it has a corresponding methylation
enzyme that protects bacterial DNA.
This is why most e. coli strains that you work with have dam- and dcm- nomenclature. This
means that these methylation enzymes have been knocked out so that they won’t affect the
ability to cut DNA isolated from these bacteria. -> important point since all organisms capable of
methylation to some degree.

7. STAR ACTIVITY: This is when your RE gets sloppy, loses specificity. The point being to
remember that these are enzymes, and to think of the sequence specificity as its substrate.
Things that can cause non-specific cleaving include things that can affect the enzyme-substrate
relationship:
(i) beyond saturating kinetics: too much of anything
(ii) change active site: buffer conditions, contaminants.

8. STOPPING REACTION: Easy. +EDTA or heat the sucker, as some enzymes are heat
sensitive. (Stick in a 65C waterbath for 10min). When in doubt, purify the DNA out, by doing
something like the phenol chloroform.
4.2 ENZYMES: Ligases/Phosphatases DAY 2
LIGATION REACTION: Ligase: enzyme that can anneal two pieces of compatible ends of DNA
together.
The important nuance to consider is that the 5’ end of your DNA ends need to be
phosphorylated.
From a compatibility point of view, if you are dealing with sticky ends (i.e. they have the
overhangs), the overhangs MUST be complementary to each other. Blunt ends can be useful in
that all blunt ends are compatible with each other. NOTE that ligation works way better for sticky
ends then for blunt ends.

T4 Ligase is considered the workhorse for this particular procedure. Conditions vary greatly
depending on the one you use, and where you bought it from, but nowadays, the procedure can
be done at room temperature in as little as 15 to 60 minutes.
** can’t directly check if ligation worked except by virtue of getting bugs to grow after
transformation.
->useful to try different insert:vector ratios. Molar ratios that often tried 1:1, 3:1, 5:1 etc. Generally
more insert is better, but every ligation is different.

CALF INTESTINAL PHOSPHATASE (CIP) Can also use alkaline phosphatase: Used to
dephosphorylate 5’ phosphates. Useful to desphorylate a cut vector, which prevents
recircularization of vector unless an insert can get in and provide the 5’ phosphases. (i.e. it's a
way to make sure that any colonies you see on the ligation plates MUST have an insert in the
multiple cloning site)
5.1 TRANSFORMATION: DNA into your host. DAY 2
The story so far… You have just done a ligation reaction where you have added a cut vector
and a fragment together. ->You want to know if the ligation worked…
FIRST: you are going to put the ligation mix into bacteria, and plate the bacteria onto ampicillin
supplemented media.

Therefore, in order for bugs to grow -> MUST have ampR. Therefore must have plasmid. But
plasmid must be circularized in order for it to get continuously and independantly replicated.
Therefore, LIGATION HAS to work to get all of this.

Is the insert in? Use the white blue system (your plates will also have X-GAL)

TRANSFORMATION:
TRANSDUCTION: all three about getting DNA into your organism.
TRANSFECTION:

Transformation is DNA into a prokaryote (except for yeast), Transduction is the use of infection
(i.e. viruses) to get DNA inside, and Transfection is DNA into a eukaryote (except for yeast).
With bugs: Two main techniques: CaCl2 and heat shock treatment and electroporation.
In both cases need to make COMPETENT cells. Cells who are primed and ready for acceptance
of DNA. Generally involves a series of growth steps so that bugs are at the just the right stage of
growth.

**In CaCl method -> competent cells have been treated with CaCl2. Essentially the membranes
are thrashed around. BOTTOM LINE: cells are delicate.
NOBODY REALLY KNOWS WHY or HOW CaCl2 + heat shock works, but it does. AND… it is
easy to do!
Ice step is believed to allow the DNA to adhere to membrane. Heat shock may make the
membrane move around more (fluid membrane). Membrane gets weaker, holes get bigger? DNA
falls in?
37 incubation allows thrashed cells to recuperate. Also gives time for plasmid to replicate so that
bugs are ampR

Electroporation: will give much higher transformation efficiencies. ZAP a current through the
bugs.
Again nobody really knows whats going on. A bit more versatile (i.e. most organisms don't have a
heat shock procedure). Microbiologists working on other bugs pseudomonads, bacilli, strept etc
etc will use this piece of machinary.
Competent cells are much easier to prepare. Procedure very quick. MORE EXPENSIVE… those
cuvettes are generally not reusable, machine cost several thousand dollars.
Different bugs use different setting, but the main idea is to vary amount of voltage applied.
Dependant on the bug, the cuvette, etc etc. All of this will translate to some magic current value
which is what causes this whole thing to work.
BECAUSE OF THIS, you need to be careful with salt content in your cells or ligation mix. Making
competent cells usually involves successive water washes.
V=IR
If you are not careful because of salt, or air pockets, etc etc.. You get something called ARCING.
This is a small explosion happening, which usually consists of a bang (to varying degrees),
cracking of the cuvette, a flash of light. General scariness but not at all dangerous.

Transfection: getting DNA into eukaryotic cells: Most common method is still to use
electroporation. ZAP the buggers!! Other alternatives include things like the Gene GUN
(Ballistic approach), and viral mechanisms.
6.1 PROTEINS: General Info DAY 3
First off, some basic things to consider that ultimately affect how you envision the behaviour of
protein in your experiments:

1. Proteins are not so simple Basic idea is that with DNA, you've got 4 different
components (nucleotides), which are all essentially quite similar anyways. WITH proteins.
20 different amino acids. All with different sorts of properties, different sorts of
charges, biochemical attributes. Consequently, proteins as a population are tough to
predict.

2. Proteins can be high maintenance: Add to that the fact that proteins can often be high
maintenance/delicate themselves, and you have an invitation for frustration. I.e. they
degrade, enzymatically go off, they aggregate, they complex, etc.

3. Protein work still has lots of toys. (Although definitely not to the extent that DNA has.
Also, one of the most powerful reagents you need – which may not be commercially
available – is a specific antibody)

4. Protein work is more fun(?): interesting to note that when pressed for opinion, most will
say that protein work tends to be more rewarding, interesting, and challenging.

We’re going to focus on two techniques, the first of which (show Nature Table of Contents) will be
the Western Blot analysis, and the second is the more fancy 2D gel electrophoresis analysis.

BE CAREFUL! keep things cold, but be sensitive to the structure of the proteins you work with –
even cold temperatures can be detrimental to your experiments.

QUANTITATION: (a few options)

- Run a gel. Compare band with preweighed amounts of standard protein (like
albumin) -> works really well, kind of labour intensive.
- USE colorimetric tests. BCA/Coomassie Blue tests works best. Easy to do, but
can be very susceptible to chemicals that are commonly used in the buffers you
store your proteins in (i.e. presence of detergents, tris, etc etc)
- ABSORBANCE at 280nm. very rough! ~1.0 O.D. = 1mg/ml of protein (but as an
example for IgG it’s closer to 1.4mg/ml)
6.2 PROTEINS: SDS PAGE. DAY 3/4
GENERAL IDEA: You have a slurry of proteins, i.e. when you lyse a particular population of cells
--> millions of proteins. YOU WANT to see if a specific protein is present in this slurry and you are
particularily interested in observing its molecular weight at the same time.

HOW DOES IT WORK?

SAMPLE PART: As proteins are quite diverse (many sizes, many shapes, many variable charge
properties – all things that will affect mobility), you need a reagent that can equalize these
parameters.

This is what your sample buffer is all about.

* has dye (so that you can see stuff)
* has glycerol (makes sample heavy so that sample will flow into wells)
* has SDS (VERY IMPORTANT) coats proteins with negative charge (now all proteins
have same charge density), and denatures proteins to uniform shape (rod-like shape).
Now all proteins have equivalent shape as well.
* has beta-mercaptoethanol or DTT (dithiothreitol) these are reducing agents. will reduce
and break disulfide bonds.

GEL PART: first you need to RUN A GEL. Often called polyacrylamide gel electrophoresis
(PAGE).
(NOTE that acrylamide is a nasty nasty neurotoxin). Acrylamide + bis-acrylamide form basis of
mesh like structure. TEMED and ammonium persulfate actually catalyse the crosslinking. More
specifically these two cause the production of free radicals resulting in covalent cross-linking of
your acrylamide fibres.
- Amounts of TEMED+ammonium persulfate dictate rate of polymerization
- Amounts of acrylamide+bis-acrylamide dictate pore sizes in mesh.

Laemmli system. (also called a discontinuous gel system) – a system to ensure you get nice tight
bands.
THE PLAYERS: STACK (low %acryl, pH6.8, Chloride ions) Cl- (leading ion)
moves faster than proteins.
RESOLVING: (set %acryl, pH8.8, Chloride ions)
RUNNING BUFFER: glycine ions. (trailing ion)

Start gel. Cl in stack and resolving gels moves quickly, glycine from buffer enters stacking gel
and moves slowly. You can envision that the Cl ions move SO fast, that there is an area of low
conductance between the Cl and glycine ions. Proteins are caught in the middle and
consequently move slowly.
When proteins reach boundary between STACK and RESOLVE, they see the resolving gel which
has smaller pore sizes making it tough for the protein to enter (this is also made worse by the fact
that the Cl ions are so far away that proteins aren't very attracted towards the positive electrode).
BUT, when the slow glycine ion reaches the interface it will become a much better glycine ion
(because of the different pH).
NET EFFECT is that all your protein sample will tighten into a sharp band at the boundary, and
then get separated according to size (proteins will be able to move into resolving because the
glycine ion is pulling it towards the positive electrode)
NOTE that because of this set up, the slowest part of the gel run is usually the stack.

STAINING: coomassie blue (0.3ug to 1ug per band) can probably see a band as faint as
100ng
silver staining 2 to 5ng (MUCH MORE SENSITIVE) very dirty procedure. If you
want figure quality data -> equipment must be ultra clean!!
ponceau red: reversible stain.
6.3 PROTEINS: Western Blot. DAY 5
WESTERN PART: essentially a procedure that allows you to probe for a specific protein using an
ANTIBODY. main idea. USE A MEMBRANE. (this is why it's called a blot).

TRANSFER: There are two main types of membrane. (i) Nitrocellulose and (ii) Nylon based
mambranes. Essentially, both rely on being hydrophobic to adhere to molecules of interest (i.e.
protein, but also DNA and RNA). Standard membranes generally have a net negative charge.
nitrocellulose is cheaper, lower binding capacity, can be brittle, not fancy.
nylon is more expensive, higher binding capacity, not brittle, lots of fancy variations out
there.

O/H picture of transfer. MOVE your proteins from the gel to the membrane by electric current.

DEVELOPING THE WESTERN: classical blot procedure: BASIC STEPS are...

- STEP 1: incubate membrane with BLOCK buffer (~2hrs) solution that covers the
rest of the membrane with generic protein (usually 5.0% milk powder or BSA)
- STEP 2: WASH membrane a bit (~5min) (usually TTBS, tween-20, tris
buffered saline)
- STEP 3: incubate membrane with PRIMARY Ab (~2hrs): Ab that is specific for
your protein of interest.
- STEP 4: WASH several times (~1/2 hr)
- STEP 5: incubate with SECONDARY Ab (~1hr): Ab that is specific for the Fc
portion (constant region) of the primary Ab. Also has an enzyme attached to it
that can convert some substrate into an observable product (=MEANS OF
DETECTION). i.e. it glows, it changes colour, etc, etc)
- STEP 6: WASH SEVERAL TIMES (~1/2 hr)
- STEP 7: ADD substrate.
TOTAL TIME: ~6hr procedure.

QUICK METHOD (millipore) Uses PVDF membrane (immobulon P) -> is highly hydrophobic
form of nylon based membrane that particularly "hates water"
THEREFORE, in theory, solutions can't actually touch membrane unless the membrane is
prewet. DRY your membrane for one hour first. Consequently, the block step is redundant, and
all incubation steps are considerably faster because you are not incubating the “whole”
membrane, you are just incubating the “parts” of the membrane that have proteins on them.
THEREFORE all steps much shorter.
PRIMARY Ab -> 40min to 1hr
SECONDARY Ab -> 20min to 30min
WASHES in seconds.

This quick method works. but you should try it first in comparison to the classical technique to
convince yourself that the technique is comparible. Some tricks associated with the technique
include a quick 20minute block step should your antibody be prone towards non-specific
binding. ALSO, you can speed up the drying process by immersing the membrane in 100%
methanol 10min, and then drying 10min.
6.4 PROTEINS: 2D gel electrophoresis. DAY 2/5
Essentially, this procedure is now way easier to pull off than even a few years ago, and is largely
due to major improvements in providing immobilized pH gradient strips (as opposed to the carrier
ampholyte gradient tube gels). As you will see, nowadays this procedure is relatively straight
forward if you have access to a set-up that makes life generally easier.

PREPARATION OF PROTEIN SAMPLES: This is arguably the most important part, and the
trickiest to advise on. In essense, you want to use a reagent recipe that result in controlled
solubilization, disaggregation, denaturation, and reduction of the proteins in the sample.
Meanwhile, you want to make sure that during this step, you don’t have too many things that can
(i) interfere with the overall ionic environment of your sample, (ii) affect the visocity of your
sample, (iii) promote protein degradation (iv) reverse the reduction process, (v) dilute the sample
too much, and of course (vi) mask the pI of your proteins. Basically any and all crap in your
sample can seriously make things messy, ugly, and not at all reproducible.

This is why, things like lysis procedure, non-ionic detergents, reducing agents, carrier
ampholytes, BUT the bottom line is to do your homework for the latest greatest preparation
protocol for your particular type of tissue.

THE RUN: First dimension. Isoelectric Focusing essentially takes advantage of settling your
protein specis at its pI where it essentially loses its net charge and therefore stays put.
Consequently, the big deal is to use a pH gradient in your first dimension. Olden days would use
a mix of carrier ampholytes within an acrylamide mix – here, the amphoyltes would migrate
according to their own prefer pI, settling at a particular place in the gel, and thereby provide their
specific buffering capabilities at a defined point in the gel. Basically you get a continuous pH
gradient. BUT, this methodology is a bit flawed because the amphlytes can drift, are often
complicated in content (therefore hard to characterize properly), and also the gel set up had a
tendency to break. In the past, this particular step may have been arguably the trickiest part.
BUT NOW:
We have immobilized pH gradient (IPGs). Here a
gradient is incorporated covalently into a
polyacrylamide gel at the time it is cast, using special
buffers (one acidic and one basic, in different ratios
along the gel).
Furthermore, casting is done on a plastic backing to
provide better reproducibility.
There is usually a rehydration step with IPGs
NOTE that IEF runs generally have very low current
values.

Second Dimension: First dimension needs to be equilibriated so that it is amenable for the
second dimension. This step, usually involves the following reagents: Buffer (happy pH), Urea +
Glycerol (elctroendosmosis), DTT (reducing agent), SDS (ionic detergent), Iodoacetamide
(alkylates thiols), Dye (so you can see the run). Once this is done, the rest is easy – basically
a normal SDS-PAGE.

DETECTION/STAIN: Radioactive > Silver Stain > Coomassie Blue

7.1 RNA: first steps DAY 3
BASICALLY, RNA work is not fun. Although it can be argued that due to its overall trendiness,
they have been a lot of recent developments in making it more fun to work with. As a molecule, it
is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because
the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get
rid of.

Oh yeah, and did I also mention that RNases are EVERYWHERE. Oh, and they don’t require
divalent cations as cofactors. If you want a sense of just how stable these suckers are, consider
the fact that they are generally unharmed by autoclaving procedures or exposure to temperatures
as high as 150C.

Because of this, you have to take the following precautions.

MUST USE ASEPTIC TECHNIQUE. Segregate all your equipment, your space, even yourself if
you have do. Different people exhibit different levels of care, which may depend a lot on your
specific locale, but a general rule is to be as careful as possible. Wear gloves; keep things cold.

USES DISPOSABLE PLASTICWARE: Essentially, there are different levels of care. There is
sterile plastic ware that you take good care of, there are RNase free grade plastic ware that you
take care of, and it’s also a good idea to use filtered tips if possible.

DECONTAMINATE OTHER EQUIPMENT FROM RNASES: There’s a couple different ways of

doing this. For instance , with glassware, you can thoroughly clean with detergent, rinse, and
then ovenbake at >250C for >4hrs. Things that are not oven friendly (please don’t put your gel
box in the oven) often use DEPC. (Diethyl pyrocarbonate)

Show O/H DEPC treated Barbie doll

DEPC is a very useful reagent for making buffers and equipment RNase free. WHAT DOES IT
DO? DEPC results in a covalent modification of nucleases rendering them inactive. Histidine
modifier – imine -> carbonate >NH >N-CO3
IN GENERAL, Work in fumehood when treating water/buffers or equipment. Usually let your
buffer + DEPC or equipment/glassware incubate at ~0.1% DEPC concentration for >12hrs.
Then leave it in fumehood O/N with venting (lid slightly ajar) SO THAT FUMES ESCAPE. Or
autoclave for faster evaporation (if possible). NOTE that technically, things are “DEPC treated”
meaning that the DEPC is no longer present.

Some nuances regarding DEPC:

- comes as a liquid/ can buy at different percentages.
- stinks. It is an organic solvent. Handy b/c you can tell its presence by its smell.
- DEPC can explode! When in contact with water creates CO2 and Ethanol. If not careful and
kept in sealed container can lead to excessive pressure buildup.
- Can't treat Tris solutions with DEPC . Tris is full of amines that DEPC will spend more of
it's time modify the wrong thing

TRICKS:
- Lots of quickie protocols out there in the web especially, although be careful.
- Chloroform also denatures Rnases, (rinse with chloroform). Can be quite harmful to
plasticware.
- Fancy reagents like “Rnase AWAY.” “RNase ZAP” Can wipe apparatus clean. Not harmful
to plastics.
- RNase inihibitors: so many classes of RNases to worry about (A, B, C, I, T1, T2...) can get
expensive.
7.2 RNA: Isolation and Purification. DAY 3
We are extracting RNA from your cheek cells using a product called Trizol.
Although this reagent is essentially a proprietory product, it is undoubtably based on the familiar
Phenol+Guanidium Thiocyanate procedure. HERE, Guanidium thiocyanate is often classed as a
denaturant although it is also considered a chaotropic salt (sucks water)
This methods works in an analogous fashion to phenol chloroform extraction except that in this
case, you also want to get your DNA to go into the phenol layer (therefore, you are left with just
RNA in the the aqueous phase). This Trizol business works because RNA is still water soluble in
a high molar guanidium thiocyanate solution whereas proteins and DNA is not. Consequently, the
insoluble components will tend to go to the organic phase. Trizol, whilst a bit old school, is
arguably still one of the best ways to get a total RNA prep from cellular material.

ALTERNATE PROCEDURES:

Another way of getting RNA (at least in eukaryotes) is to utilize the fact that your RNA resides in
the cytoplasm whilst the DNA resides in the nucleus (at least for eukaryotes). Consequently, an
option is to first treat the cell with a "gentle" detergent to lyse the cell membrane but leave the
nuclear membrane intact. Examples of common detergents used for this purpose are Triton X-
100 and NP-40 (these two are almost identical). NOTE: The biochemistry and behaviour of
detergents is very complicated. CONSEQUENTLY when dealing with a detergent, it is a good
idea to follow the procedures given rather than playing around too much. Detergents have many
attributes that affect their effectiveness. Dependant on their existence as free form particles or
micelle complexes (kinda like a vesicle). Detergents forming micelles don't work as well and
micelle formation is very sensitive to both temperature and concentration effects which vary
enormously from detergent to detergent.

CHECKING STUFF OUT ON A GEL:

Running RNA on a gel has a few extra considerations to note.
Firstly, most systems use a MOPS, MES buffer (getting away
from Tris). In addition, RNA is usually treated with a
denaturant step (i.e. + formaldehyde, or use DMSO +
glyoxal), since RNA loves to form tertiary structures.
NOTE that total RNA preps look decidedly unspectacular.
Normally, you see two or three very bright bands
corresponding to ribosomal RNA (especially 28S/18S which
run at around 5kb and 1.7kb respectively), with lighter bands
throughout (mRNA spec)
7.3 RNA: Gene Expression. DAY 3

MORE STUFF ON RNA:

Total RNA is ~ %80 rRNA
%15 tRNA
<5% mRNA

most people concerned about mRNA (although some study rRNA as an evolutionary marker
since the rRNA moelcules are very conserved from species to species -> use it to gage
evolutionary trees).
Generally, the cleaner the mRNA prep the easier your life is gonna be. BUT getting purified
mRNA sample requires additional steps.

NOTE: Almost all mRNA have poly A tails, which is a very useful characteristic that many mRNA
techniques take advantage of...

5’----------------------------AAAAAAAAAAAAA 3’

Problem is that in this case, RNase degradation is an even bigger concern, since these
nucleases tend to chew the ends of RNA first. Point is that whilst the majority of your RNA is fine,
you may lose the one region that is important for many subsequent steps.
Examples of using the polyA tail.
YOU can use oligo dT affinity chromatography to purify your mRNA.
Can use the polyA region as a primer binding site for reverse transcriptase or PCR experiments.

Reverse transcriptase: some specifics:

NOTE that the procedure itself is very straightforward. Difficulties arise primarily because of the
RNA prep.
The enzyme: Number of commercially variants available, that are usually derived from AMV
(avian myeloblastosis virus), MMLV (moloney murine leukemia virus), or HIV (human
immunodeficiency virus).

RTs generally contain some combination of four basic functions: (1) RNA-dependent DNA
polymerase, (2) hybrid dependant ribonuclease (RNaseH), (3) end point hairpin loop
formation, and (4) DNA dependant DNA polymerase.

For EST (expressed sequence tag) or mRNA work: Oligo dT primer usually a minimum of 12
nucleotides in length. Commonly in the 12-20 nucleotide range.

TWO STEP vs ONE STEP RT-PCR:

two step is generally more reliable as you have the option of tweaking your PCR parameters (will
talk later). i.e. you can make your PCR go at its optimal best, because the reaction can be fine
tuned independantly. Sometimes you have to do two step because the RT you use prefers Mn2+
as a cofactor. More likelihood for cross contamination since there are more steps - this can be a
problem if your mRNA is in very low amounts and the sensitivity of your assay is high.

one step is quicker, less work BUT also possibly less reliable because your PCR reaction
conditions are constrained by some of the conditions used in your reverse transcriptase assay.
i.e. cofactor amounts stay the same. Affect of your PCR primers in RT assay, etc etc etc. One
step works because the two enzymes (RT and the heat stable DNA pol) work and can be
differentially activated at different temperatures.
8.1 POLYMERASE CHAIN REACTION - The basics DAY 1
PCR (polymerase chain reaction) is an excellent example of illustrating how it's often the most
simple and elegant ideas that really propel science. A bit of history :Kary Mullis is the principle
investigator behind the techniques. Interesting (slightly eccentric fella) who won a nobel prize in
1993 for the technique (actually shared the nobel that year with our very own Michael Smith.

Basic premise.
Working with DNA. If you want to manipulate it, or even see it --> needs LOTS of it.
PCR is an attempt to amplify DNA in a test tube environment.

EXCEPT that this amplification occurs via relpication in the natural sense, and replication is a little
on the complicated side (in terms of getting it to work in an in vitro setting). So, Dr Mullis
essentially tried to MacGyver his way through the experiment.
- POINT ONE: replication requires open or single stranded DNA. Usually
accomplished by many enzymes that unwind DNA (such as helicases). Screw
the enzyme - Let's use HEAT to open up our DNA.
- POINT TWO: replication also needs a primase enzyme to make primer for the
polymerase. BUT, you can make your own! Buy oligo's.
- POINT THREE: with the use of a DNA primer system, you don’t need DNA pol I.
- POINT FOUR: with a forward and reverse primer system, don’t need LIGASE.
- POINT FIVE: with heat globally opening your DNA, you don’t need
TOPOISOMERASES either.
- POINT SIX: You can get this to work with only a workshorse DNA polymerase
(i.e. one enzyme).
HOWEVER, we do have one problem: this high temperature will basically knacker out any protein
structures,including our polymerase. TO get around this, let's use a polymerase from a bug that
grows in high temperatures (thermophile). i.e. this will be a heat stable polymerase.

HUGE ADVANTAGE #1: PCR is elegantly simple, and extremely forgiving procedure.
8.1 CONTINUED. DAY 1

PCR cycle is usually composed of three steps: (i) denaturation, (ii) annealing, and (iii) elongation.
Essentially, each cycle is responsible for doubling the amount of target DNA. A cycle can take
anywhere from 1.5 minutes to 5 minutes long, meaning that after 30 cycles, you have the
potential to produce ~1000000000 molecules of amplified product from one molecule of template.

HUGE ADVANTAGE #2: PCR gives you data.

8.1 CONTINUED. DAY 1
ALSO, individuals over the last 15 years have been quite creative with the technique to do some
very cool things. For example:

*site-directed mutagenesis
*production of restriction endonuclease site for convenient cloning
*production of single and double strand product for sequencing protocols
*quantitation of rare DNA
*amplification of partial cDNA sequences
*quantitation of mRNA expression
*differential display of mRNA by PCR
*random amplified polymorphic DNA (RAPD)
*cloning dinosaurs from dinosaur blood found in fossilized mosquitos trapped in amber

HUGE ADVANTAGE #3: PCR is very versatile procedure with many possible uses.
8.2 PCR - the specifics. DAY 1
WHAT THE BIG PICTURE ACTUALLY LOOKS LIKE.
NOT SO BASICALLY:

and, as if this isn’t bad enough, the kinetics of each of these arrows change with each cycle since
the amounts will change after each round of amplication.

Yikes! (and this doesn’t even include the reverse transcriptase stuff)

AND OF COURSE, the other big thing to wrap your head around is to understand exactly what it
is you are trying to do with your PCR reaction. Here there are a few main things this line of
questioning can fit under.

i. Yes or no, please.

ii. I’m very much into amounts.
iii. I need my product for this other thing.
iv. Sorry to tell you this, but I have very little to start with.
v. Hey get this! I have no idea what I’m looking for!
8.2 PCR CONTINUED DAY 1
O.K. WHAT ARE THE SPECIFICS?
1. General PCR buffer: salt + buffer + detergent.
FOR EXAMPLE: 50mM KCl, 10mM Tris-Cl @ pH9.0, 0.01% Triton X-100
This is simply a recipe that makes the polymerase happy.

2. Need Template DNA to amplify. This is the DNA you will start with. ~1.0ug mammalian
genomic DNA/10ul or 0.1ng plasmid DNA/10ul use 10ul per reaction. These amounts, of course,
are variable and ultimately have a lot to do with the primers you use. THE PURER THE BETTER,
but PCR is quite forgiving.

3. Need primers

(Primers tend to be the most strategic portion of designing a PCR reaction. SO MUCH SO, that
there is lots of free and commercial software that you can use to help design primers). In this
respect, a good representative list of what’s available can be found at the following URL:

[Link]
In fact, this resource is useful to browse through a huge variety of different software, specifically
scripted for a particular use.

HOWEVER, if we stick to the primer design side of things:

WHAT appears to be the most commonly used, and therefore perhaps most trusted program is
the PRIMER3 software. However, different people have different preferences (PRIMER3 doesn’t
have the most user friendly interface for instance).

To find PRIMER3, google it – you’ll come across a variety of different web interfaces for the
algorhythm.

ON THE OTHER HAND, it’s worth knowing what these programs are doing from a conceptual
sense. Here, what I mean, is that there is enormous value is understanding the many aspects
that influence strategic primer design. This type of information is especially useful in the context
of designing primers where geography is set (i.e. making a product for restriction cloning). Here,
your primers are restricted to very specific locations, so the other considerations become much
more important.

SO, what are these considerations? LISTED IN NO PARTICULAR ORDER:

1. 3’ terminal position is crucial to prevent mispriming, particularly since this is the point of
contact with your polymerase. Consequently, it is advantageous to have G’s and C’s
near this end (often called GC clamping).

2. Primers should avoid long runs of single nucleotides (especially A’s and T’s), this can
result in a slipping effect. In a similar manner, avoid direct multiple repeats in the primer
itself, which is often a common place for secondary structure interactions.

3. No intramolecular homology - > secondary structure which can interfere with template
annealing, or completely block the polymerase from starting.

4. No Intermolecular homology -> kinetically more favourable for two primers to anneal to
each other (given their saturating amounts). This is especially prevalent when looking at
the 3’ ends of your primers.
8.2 PCR CONTINUED DAY 1
5. DNA primers are categorically between 15 and 30 nucleotides, GC content at ~50% but
you basically want to aim for a calculated melting temperature of approximately 55C to
60C. Manual way that you can arrive at this number includes using:
o o
Tm = (A+T)2 C + (G+C)4 C

By increasing the primer length, you are also allowed to increase the annealing
temperature and thus obtain a higher specificity in your PCR reaction. On the other hand,
by increasing the primer length without increasing the annealing temperature
correspondingly, you allow for more wobble (that is primer-template mismatch)

6. All primers (and possibly probes) in the process should have similar Tm’s. This is
because there is one single annealing step in the process, and therefore this should be
amenable to all primers used.

7. Primers are normally used at excess, which normally falls within the 100 to 500nM range.

8. AND OBVIOUSLY, choose the sequence of your primer carefully. Mismatches will affect
specific binding, and if the mismatch occurs at the 3’ end, may affect the ability of the
polymerase to read.

9. Make sure your primer doesn’t bind elsewhere, or has the potential to bind elsewhere.
Use BLAST to query available databases for this problem, or use software like PUNS.

10. Consider post-replication variants when possible - i.e. things like splice variants.

11. Deliberate mismatches (i.e. for introducing restriction sites, or point mutations) should
always occur at the 5’ end (away from the polymerase).

12. Distance between forward and reverse primers will need to coordinate with polymerase
used. i.e. how big is that amplified product? GENERALLY SPEAKING: <3kb = easy
whereas >10kb can be very challenging. For real time experiments, the smaller the
better (this is why they are often between 50 and 100bp in size).

13. Please tell me you’re not doing an experiment with degenerate or RAPD primers,
unless, of course, you have no choice.

Alright. Go buy some primers now. They cost about 40c to 50c a base for a 40nmole prep. (i.e.
20mer will only cost about $10).

4. need dNTPs ~0.2mM mix. don't mess around -> enough to make 12.5ug of product!!!!!

5. Heat Stable DNA Polymerase: Enzyme usually comes in a stock of about 2-5Units/ul. need
~2.5units per reaction. NOTE, there used to be two main subtypes: Taq (cheaper + higher error
rate), and Vent (more expensive + lower error rate). Take care to note nuances of the polymerase
you use.
i.e. - Taq can do this wierd thing where an ATP is added to the 3' ends of the PCR product.
- Some polymerases don’t like SYBR green.
- Some polymerases better at incorporating nucleotide analogs.
8.2 PCR CONTINUED DAY 1
THE POLYMERASE SO MANY CHOICES
Really really confusing stuff. If I had to pick an analogy. choosing the appropriate polymerase
is not unlike buying your first house. In other words, there are a multitude of factors that you
can take into consideration when selecting your enzyme.

KEEP IN MIND HOWEVER, that because of the large selection of different polymerases, a
company will have no problems with giving you a free sample of said product for you to try out.
Here is a general table of some of the products available and why you’re thinking about these
particular traits. (NOTE that these values were access independantly by Stephen A. Bustin and
Tania Nolan from “A-Z of Quantitative PCR”)

Half life Elongation Processivity Fidelity dUTP 5’-3' 3’-5’

Enzyme 95°C (h) rate (nt/s) (nt) o.k. exo exo

KOD HiFi 12 100-130 >300 +++++ ? N Y

Pfu 6-18 25 10-20 ++++ N N Y
Tth 0.3 25-33 30-40 + Y? Y N
Pwo ? 40-50 40 +++ N N Y
Tgo 2 ? ? +++++ Y? N Y
Vent 6.7 67 10 ++ N N Y
Deep Vent 23 23 <20 +++ N N Y
9°Nm 7.7 ? ? ? N 5%
Taq 1.6 60 150 + Y Y N
Tfl 0.6 40 50-60 + Y Y N

1. Half Life: generally accesses the heat stability of said enzyme. Since the average PCR
reaction will likely take no more than 4 hours, the values stated here are really not that
important in the grand scheme of things.
2. Elongation rate: This is more of a consideration when dealing with amplicons of
significant size. i.e. not in the case of real time experiments where your amplified product
is quite small. Current literature suggests maximum size of fragment that can be
produced efficiently is about 40kb in size (as stated by BioRad’s iProof).
3. Processivity: This term applies to the general idea of how long can the polymerase hold
on for. Because these polymerases are working at inordinately high temperatures, you
have a situation where they are literally continually falling on and off. High Processivity is
most advantageous with large fragment sizes.
4. Fidelity is key when producing amplicons that ultimately have an auterior destination (i.e.
for cloning). In general numbers will hover around 1x10-4 to 1.6x10-6 errors per
nucleotide. (NOTE that native Taq can have an error rate as high as 1 in 400). However
also keep in mind that if you are doing a reaction with high numbers of cycle, the chance
of a mutation possibly happening within the primer binding sequence (and most notably
at it’s 3’ end) could be an issue.
5. 5’-3’ exonuclease activity: Absolutely crucial for TaqMan probes. Functions overall with
regards to excision repair within a cell (as well as to deal with the Okazaki fragments),
which is not so much an issue here. Therefore, logistically, it is an attribute that slows the
polymerase down.
6. 3’-5’ exonuclease activity: This is primarily associated with proof reading ability.
7. Does it like dUTP. Can be an issue when dealing with amplicon contamination.
OTHER ISSUES NOT LISTED ABOVE:
8. RT activity: curiously enough, you’ll find that some polymerases can also function as
reverse transcriptases. This is something that is occassionally taken advantage of in one
8.2 PCR CONTINUED DAY 1
step reverse transcriptase PCR procedures (will touch on this later, but the main one
here is Tth).
9. Compatibility with SYBR reagents.
10. A overhang nuance. Some polymerases have the propensity to finish off each 3’ end of
the amplicon with an additional A (resulting in a single A overhang).

Beyond the above, the crazy thing is that all of these “attributes” (especially when described by
the company pushing the product) can vary significantly depending on the components within the
reaction buffer. In addition, comparitive studies advertised by companies to make claims are
always done under non-standardize conditions making true comparisons practically impossible.
Add to that, many proprietary versions of said polymerase (mutated, genetically engineered etc),
which have altered abilities tends to only complicate the analysis further (for instance KlenTaq is
a Taq derivative with the 5’ exo region has been “toughed up”, for better performance overall).

Chemical Name Final Concentration

BSA No inhibition
NH4 No inhibition
NP-40 No inhibition
Propidiurn Iodide No inhibition
Spermidine No inhibition
Ca 2+ > 3,5 mM
Chloroform >50 mM
Dimethylformamide >50 mM
DMSO >10%
DTT > 1 mM
EDTA >50mM
Ferric ion >10uM
Formamide >50 mM
Hemoglohin/heme heme will interfere with PCR
NaCI >50mM
Phenol >50 mM
KCI >50mM
SDS >50mM
Siliconized tubes inhibition
TritonX-l00 inhibition

Mg2+ concentrations: Still one of the most important attributes affecting polymerase (as well as
template/primer). +1A at high Mg2+

In any event, the following is some useful info regarding some specific polymerases (almost like
personals ads)
1. Taq (thermus aquaticus)
Not looking for surprises or flashiness. Just something trustworthy and predictable. Must
appreciate some eccentric nucleotides. Note Fidelity is great at certain pH, which may not
correspond specifically to best processivity (at high pH’s).
2. KlenTaq: (Stoffel Fragment)
Is your 5’ exo region thermally tough? If so call on me, and we’ll do wonders with TaqMan
systems, and general robustness overall.
3. Tth: (Thermus thermophilus)
Are you into organics or RNA? Not a problem, give me a call.
4. Tfl: (Thermus Flavus)
Tth too wussy for you. Then I’m your polymerase.
5. Pfu: (Pyrococcus furiosus)
I may be slow but if its longevity and fidelity that counts, then give me a buzz – I’ll be waiting.
6. Vent: (Thermococcus litoralis)
Taq too boring for you, but the others are still a bit too crazy, even though fidelity is important to
you. Vent is probably what you’re looking for.
8.2 PCR CONTINUED DAY 1
7. Deep Vent: (Pyrococcus species)
Hot damn! I’m super stable and extremely low maintenance (love formamide and DMSO). Also
very good fidelity, but still with high elongation rates.

pTaq = I’m free baby, all free baby.

iProof = call me top of the line baby, top of the line baby (but not compatible with SYBR)

6. Thermal Cycler: can range from CAN$2000 to over CAN$90,000. Depends on capabilities,
reliability, reproducibility, and precision (as well as special features like the optical unit on a real
time PCR machine)

7. The Cycle: Generally speaking:

Start with something called a “cold” or “hot” start. 95C for an extended period of time (2-
4minutes).
These are tricks to optimize the first round of the PCR reaction which is crucial to avoid
diluting the amplification process. i.e. sample prep at room temperature can lead to non-specific
priming events, under the auspices of a functional (albeit slow) polymerase.

Denaturation STEP: 95C 15sec to 1minute. Time more dependant on size of template, (i.e.
genomic DNA may want to give more time to this step) but particularly is dependant on size of
amplicon.

Annealing Step: Temperature is very important. Can be specific (dependant on Tm calculation),

can be generally determined (i.e. GC <50% use 55C, if >50% use 60C). Usually about 15sec to
1 minute
NOTE that some machines allow the option of doing a gradient within your multiwell sample
set up, so that you can check multiple annealing temperatures in a single experiment.
NOTE that some machines will allow you to program a gradient ramp within the annealing
step, should you be stuck with a primer set of very different Tm (i.e. the annealing step goes from
high to low)

ALSO: Touchdown PCR, which is where annealing temperature will get lower as the number of
cycles increase. Idea being that this facilitates specific priming initially, but as amplified product is
generated, the stringency of the annealing temperature can be relaxed to allow greater
amplification efficiency.

Elongation Step: Temperature is usually around 72C (Favourite for Taq), time will depend on
elongation rates, but can vary between 30sec and 3-5minutes.

TWO-TEMPERATURE PCR: this is where the annealing and elongation steps are combined into
one step. This method works optimally when primers have a Tm > 60C. Particularily useful with
Taq derivatives since this polymerase is still very active at 60C.

NOTE: that total time for the PCR reaction also includes RAMP time which is the
speed at which the temperature changes can occur.
8.3 PCR – Troubleshooting DAY 1
Generally, PCR is a standardized procedure but due to differences in primer and template,
evitably you may come across a PCR that won't go. SO,.. when you're doing a particular PCR for
the first time, it is worth figuring out the best conditions.

WHAT are the conditions to play around with? BASICALLY, this has to do with things that affect
the ability of the primers to go to the right place (hybridization paramaters) – it’s a kinetic thing.

OPTION ONE: MgCl2:: VERY COMMON thing to play around with, although exact reasons why
are a little vague. In essense, Mg ions seems to do a number of things.
- it binds DNA: may affect primer/template interactions.
- it binds the DNA polymerase -> required as a cofactor
- it influences the DNA polymerase’s ability to interact with primer/template
sequences.
BOTTOM LINE is that more Mg = less stringency in binding.
DO a titration. generally people will check between 1 and 6mM final concentration.

OPTION TWO: Kinetic parameters. Template, Primer, Enzyme concentrations. What’s the
likelihood of things interacting appropriately, not appropriately, not at all, etc. Annealing
[Link] a common option, especially with machines that can easily provide
temperature gradients in the PCR.

OPTION THREE: Template quality. Said this before, but why introduce caveats by having a
sample that is not high quality. That being said, one of PCR’s great appeal is that with a well
designed strategy, you may not need to clean your template up.

OPTION FOUR: BONUS REAGENTS:

+/- DMSO denaturant ability. good at keeping GC rich template/primer strands from forming
secondary structures. Doesn't seem to generally affect the reaction. Most people will include it
regardless (use at 5%)

+/- glycerol increases apparent concentration of primer/template mix. Therefore may help in
getting good primer/template interactions at high temperatures. (use @ 10%)

AND... OPTIMIZATION of the first round of amplification. (IMPORTANT)

General idea is that whilst you are setting up your reaction, the taq pol. may start replication when
the primer is binding at an inappropriate site. This will lead to "pseudo" bands and a diluting out
effect of your real template. SO,.. there are a couple tricks to optimize your first cycle.
-"hot start" + polymerase after first denaturation and annealing step -> go to RT.
-ice cooling after annealing temperature -> go to ice temp.
-taqstart antibody. Includes addition of taq antibody which will get denatured upon 95C step.

FINALLY CHECK THAT THERMAL CYCLER IS WORKING. TEMPERATURE NEEDS TO BE

SPOT ON FOR THIS WHOLE THING TO WORK PROPERLY.
8.4 REAL TIME PCR. DAY 4
This is, essentially, the use of chemistry and optics to detect and evaluate production of
PCR products in real time. i.e. conventional PCR is based on end-point analysis -> you look at
a band on a gel, you look at your data at one point in your reaction which happens to be when it’s
all finished.
WHICH is flawed in principle, if quantitation is something you are interested in. With endpoint
data representation you are looking at a tired reaction (enzymes going off, reagents becoming
limiting etc).

1600000000
1400000000
AMOUNT OF DNA 1200000000
1000000000
800000000
600000000
400000000
200000000
0
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER

So, real time enables you to watch the kinetics of the reaction, which ultimately means your ability
to quantify the reaction is significantly improved.

HOW do you do it: FANCY CHEMISTRY:

Two main types:

SYBR systems: a reagent that selectively binds to
double stranded DNA, and flouresces under this
interaction. CHEAPEST way – by far the most
common.
ALSO extremely useful for melt curves, which provide
a mechanism to look at your amplification products.

TAQMAN systems: a two component system that

relies on something known as the FRET (Fluoresence
Resonance Energy Transfer) phenomenon. Here,
there are reagents that can donate energy (R) at
specific wavelengths when excited, AS WELL as
acceptor reagents that can quench (Q) this energy if
located in close enough proximity. i.e. the two
chemistries are located on the same TAQMAN probe.

Because of Taq’s 5’ exonuclease activity, one can

separate the two components during amplification.
Therefore monitor PCR reaction as a consequence of
TAQMAN probe degradation -> Emitter is released
from quencher, so that energy can now be detected.
Because there are a variety of different FRET systems,
available, TAQMAN methods are useful for
multiplexing experiments (especially in Single
Nucleotide Polymorphism determination

Overall advantages, include increase sensitivity, increased speed. Often products being
observed are deliberately small, since we are more interested in an effective PCR reaction, that
the band of DNA at the end of the procedure.
8.4 CONTINUED DAY 4

Quantitation: THE REALITY.

So what’s the problem? The problem is that

at its heart reverse transcriptase Real Time
PCR is all about quantitation. In fact, it is
currently considered the gold standard
technique for answering this type of biological
question.

The reality, however, is that your final readout

value, whilst theorectically provides a
quantitative reflection of your answer, is
actually derived from a lot of steps, where at
each point, that confidence in that correlation
goes down.

If you take the following diagram as an

example, you can see multiple points where
things can unduly affect your final data point
(and this isn’t even considering the actual
experiment on the tissue source itself!).

For example, step 5 questions the efficiency

of the Reverse Transcriptase itself. In
experiments done with internal RNA controls,
it has been shown that efficiency values of
commonly available RTs can fit anywhere
between 14 and 85% (!!!). This means that
already, your cDNA amounts will not reflect
your actual RNA amounts (Invitrogen
Platinum, by the way, won this contest).

A QUESTION OF PRECISION VS
ACCURACY...

Then, there is the question of just what is an

appropriate control to generate a standard
curve.
- A housekeeping gene?
- Cell numbers?
- RNA controls? (univeral, T7
prepared)
- synthetic Armoured RNA
controls?
- cDNAs / sense strand oligos?
APPENDIX A: Replication. PRE-READING
(Use magnetic board)
dancers, the two lines are going whereas the phosphate group is
in opposite directions. This prominent, overbearing even.
picture should help: This turns out to be a crucial
factor because replication is
carried out by the activities of a
variety of different enzymes
To begin with, we’ll start with a which all function by focusing on
chicken scratch drawing of a one DNA end or another or
DNA molecule, which you know both.
is double stranded. My poor
pathetic attempt at illustration is So now, the picture looks like
therefore going to look like this: Your DNA strands are doing this:
something very similar in a
chemical sense. The difference,
of course, is that instead of
dancers, you have your choice
of four nucleotides.
It should also be pointed out
You also know that each strand Furthermore, like the situation of
that DNA is not really like this
of DNA is composed of building left hands versus right hands,
flat goofy looking cartoon. As
blocks called nucleotides, and the ends of the DNA strands are
mentioned in a previous
that these nucleotides are also different. One end is known
chapter, the two DNA strands
always interacting in a as the 3’ (pronounced 3 prime)
are actually intertwined around
complementary manner. For end and the other is known as
each other in a rather pretty
example, A’s are always with the 5’ end. To the layman, these
helical fashion. This is where
T’s, C’s are always with G’s, rather stoic terms are an
the two strands are wound
Beavis is always with Butthead, unfortunate consequence of
around each other, sort of like
etc etc etc. Let’s draw them in chemical labeling. So now, our
two elastic strings twisted and
like so: picture should look like this:
coiled together. Sort of like this:

Now that the stage is set, it’s

What you haven’t been told at time to introduce the proteins or
this point is that chemically I should reemphasis that the 3’ the enzymes, which are
speaking, the two strands are and 5’ ends are very different responsible for the actual
going in opposite directions. The from each other. To be more process of replication. Enzyme
correct term for this is actually specific, we say that they are is just a fancy word for a protein
known as anti-parallelism. To chemically distinct from each that is able to facilitate a
denote this, I’ll draw some other. They are as different from chemical process. What I’ll do
arrowheads on the DNA each other as apples and here is to focus on terminology
strands: oranges. In fact the 3’ end is associated with a simple
composed of a hydroxide group organism like the bacteria, e.
and the 5’ end is composed of coli. However, all organisms,
something known as a even those as complicated as
phosphate group. These groups humans, do more or less the
look a little like this: same thing when it comes to
doubling their DNA — the
principle difference being that
unfortunately, the enzymes
Although, this may seem a little have difference names and
confusing at first, try to picture Hopefully, it’s easy to see that labels.
two lines of square dancers they are indeed distinct from
facing each other. In this each other —even more so than That aside, the first enzyme for
circumstance, you notice that apples and oranges. The replication in e. coli that we
when focusing on the left or hydroxide group being should introduce is, of course,
right hands of the row of comparatively small and meek, the most important enzyme in
the entire process. In e. coli, this
enzyme is called DNA whims of the replication Rule number one states that all
polymerase III (or DNA pol III for machinary. DNA polymerases function by
short), and is essentially the one adding nucleotides to the 3’ end
that is responsible for the actual Curiously, the DNA pol III, which of the DNA strand. What this
business of making more DNA. after the unwinding event, can means exactly is that a DNA
If this entire exercise was now interact with the DNA strand can be extended by the
analogous to a movie, then this molecules, does so whilst addition of new A’s, T’s, C’s or
enzyme is the marquee player. attached to a bunch of other G’s. However, the new
It is the Tom Cruise, the Julia enzymes. This attachment is a nucleotides can only be added
Roberts, the proverbial bread little like a bunch of buddies to one particular end, namely
and butter of replication. It is, hanging out together. The the 3’ hydroxide group. This is a
quite simply, the star of the complex actually looks a little molecular restraint in that the
entire process. Instead of like this: DNA polymerase can only join
drawing a picture of Tom Cruise nucleotides via this smallish
or a picture of Julia Roberts, I chemical group. This rule can
think a picture like this should be drawn out like this:
suffice:

You’ll notice it has the

following... (i) two DNA
Problem is, if we were to draw
polymerase III’s: which kind of Rule number two states that all
this enzyme to scale with a
makes sense given the fact that DNA polymerases require a
helical DNA molecule (like this),
there are two strands of DNA primer to function properly. This
that need to be copied; (ii) one is probably the most challenging
helicase molecule: which also concept that needs to be
sort of makes sense, because addressed. If you get through
as this replication complex is this, then you consider yourself
doing its thing along the DNA home free.
you’ll notice that the DNA pol III
molecule, wouldn’t it be handy
is actually too big to get inside
to have the built-in ability of To simplify the notion of a
the DNA strands. It can’t go
opening up the DNA molecule primer, let’s look at a single
about its business of copying
as it moves along; and (iii) one strand of DNA, complete with its
the DNA, because the strands
new enzyme which is known in 5’ and 3’ ends. It should look a
are all coiled up in the helical
e. coli as the primase. However, bit like this:
structure. In other words, there
the purpose of the primase
is a serious issue of
molecule is a little complicated
accessibility. Even our star
and so to fully comprehend the
enzyme, despite its importance,
role of this enzyme, we need to
can’t do its job without access to Now according to rule number
switch gears a little and tell you
the molecules of DNA it wants one, a DNA polymerase can
a bit more about the DNA pol III
to copy. Consequently, the extend this single strand chain
molecule.
enzyme that inevitably has to but only by adding nucleotides
act first is one that is to the 3’ end. In effect, you can
What actually needs to be done,
responsible for opening up the argue that all of the relevant
is for us to go over a few
DNA strand. This enzyme is chemical groups are present for
mechanisms that all DNA
known as a helicase, and its making more DNA. However,
polymerases seem to use. In
role is to essentially unwind the the problem lies in the fact that
fact, it’s apparent that every
DNA molecule, which would under these circumstances, the
DNA polymerase that has been
look like this: DNA polymerase doesn’t
discovered on this planet:
actually know what to add. How
does it know, whether to add an
A, a T, a C or a G? It can’t
exactly be a random event,
because replication is all about
The net effect being the making sure cells receives an
In fact they all (without
production of a "bubble" of exception) seem to follow a two identical copy of the DNA code.
opening where the two DNA basic rules.
strands are pried apart and are Take the following picture:
subsequently accessible to the
 So,.. the small sequence of
nucleotides that has been
circled here...

Under this layout, it should be

clear that now, the DNA
polymerase has the required 3’
group, AND it also has a ... which makes an area of
template to read and ascertain double strandedness is However, it’s wise to pause here
what those nucleotides should technically known as a primer. for a second, because you have
be. For instance, if the With this all sorted out, hopefully to understand that whilst this top
nucleotide in the opposite strand the rule about requiring this strand is being replicated, the
is a G, then the DNA primer makes a little more lower strand is also being
polymerase knows it should add sense, and you can probably worked on simultaneously
a C. If the nucleotide in the guess that the enzyme called (There are two DNA polymerase
opposite strand is a T, then the the primase may have III’s attached together afterall).
DNA polymerase knows it something to do with this The lower strand is actually a bit
should add a A. Hopefully, at nuance. messier for reasons that will
this point, you’ll at least agree
become clearer as we proceed
with the following statement. A Which turns out to be exactly in this discussion.
DNA polymerase can not do what this primase enzyme is all
anything with a single strand of about. In a nutshell, it is an Basically, the primase enzyme
DNA. True, it has the right enzyme capable of making a will also go about preparing a
chemistry, but in effect, it does short sequence of nucleic acids primer for the lower strands.
not have the template or which functions as a primer. A However, if we draw this primer
instructions needed to define key point that needs to be and label the ends in the anti-
how the chain is extended. emphasized, however, is that parallel manner, you can
this primer is made up of RNA, hopefully see a logistical
If we redraw the picture. Say which if you recall, is a molecule problem in this set-up. Take a
like this: that is very similar to DNA in look at the following picture, and
that it is also composed of the see if you can find the problem
representative four nucleotide (remember, the DNA
code. This is actually due to a polymerases, the helicase and
biological technicality whereby it the primase all move as a single
is possible to make a unit in one direction, and
What you’ll notice are two complementary strand of RNA remember that all DNA
strands of DNA, one long and without the use of a primer polymerases must add to the 3’
one short. You’ll also notice that (hmmm, think about this for a end):
the strands are anti-parallel as second). Taken together, the
discussed earlier. If you focus function of the primase should
on the arrowhead, you’ll find end up looking like this:
yourself focusing on a perfectly
situated 3’ group. Here is the
end of a DNA strand that is
chemically ready to have
nucleotides attached.
Furthermore, it is also a 3’ end
that is located where a template
is present on the opposite If you’ve been following along,
then hopefully you can see that Do you see the problem? Do
strand. In other words, you see a problem with the
everything is in place. The right replication from this RNA primer
can proceed in a manner that direction of the primer? Do you
chemistry, and a means for see that the 3’ end of the lower
instructing which nucleotides to can be drawn like this:
primer is facing the wrong
add. Again, taken at the direction?
simplest level, we can conclude
that in order for a DNA This is obviously a problem, and
polymerase to do its thing, it it turns out that in order to
needs an area of double overcome it, the DNA
strandedness. polymerase will still add
nucleotides to the 3’ end, but
can only do so for a short as a fish with sharp teeth. This
distance. To keep it simple, enzyme is special in that, in a
think of it as being able to nutshell, it is responsible for
replicate as far as the enzyme is dealing with the RNA. In a
big, which should look a little nutshell, its job is to somehow
like this: replace it with DNA. In a
nutshell, I’ll draw it like this:

Now, if you take all of this into

consideration, you come up with
the following mechanism. DNA
DNA polymerase actually has polymerase I will come in on our
two distinct functions. Firstly, as replication picture, and zone in
its name implies, it is a DNA on a nick in the strands. Once
Unfortunately, this doesn’t polymerase, meaning that it is there, it will begin chewing on
inherently solve the direction capable of extending the DNA the 5’ end, which should look a
problem, so what ends up chain, but in doing so must bit like this:
happening, is that with this follow the same two rules that
lower strand, the primase has to govern these enzymes. In other
continually make a primer, and words, it must add nucleotides
the DNA polymerase III has to to the 3’ end and it must use a
continually replicate a little bit. In primer as a springboard.
the end, it should look like this: Ironically, it is a shitty DNA
polymerase. Whereas DNA Don’t forget that this enzyme is
polymerase III can replicate for also a DNA polymerase, and if
several hundred nucleotides, you look at the other side of the
DNA polymerase I has difficulty nick, you will hopefully realize
getting past a few dozen. that there is this beautiful 3’ end
ready for action. This beautiful
Secondly, DNA polymerase I is 3’ end is right here:
also an exonuclease. This
means it’s capable of degrading
or chewing up nucleotides.
Which is another reason why I
The difference in how each drew a fish with teeth. And not
strand gets copied is reflected in only does it chew stuff up, it
why some people call them the does so in a fairly specific
leading and lagging strands of manner. To begin with, it likes to
replication. One strand is start at areas, which are termed
Let’s say that the fish’s ass
obviously fairly straight forward as nicks in the DNA. In our
happens to contain the DNA
whereas the other is quite picture, this is where the nicks
polymerase function. What
labour intensive. would be:
therefore happens is that DNA
polymerase I will start
Anyhow, after this is all said and replicating from that 3’ end,
done, hopefully, you’ll agree which incidentally fills up the
with the following statement. gap that was created by the
That is, we have finally doubled exonuclease activity. This
or copied our genetic sequence. should nicely demonstrate how
However, it should also be clear DNA pol I achieves its function
that the whole thing is a bit of replacing the RNA with DNA.
messy. For instance, there are This whole step should kind of
bits of RNA everywhere, and the look like this:
lagging strand is composed of Furthermore, this exonuclease
pieces. To address these is picky in that it always chews
problems, we have to introduce from the 5’ end. Basically it is
a few more enzymes. gunning for that big phosphate
group. So that you don’t forget
The first of which is DNA this, I’ve drawn this picture to
polymerase I, which I will draw help you visualize this:
 will notice that each of the DNA band and pry them open. It
Hopefully, this puts the sequences is derived from one should make a little bubble and
shittiness of this DNA pol I in old strand and one newly should look a little like this:
perspective. It's quite synthesized strand. Because of
biologically pretty because, I this, replication is often termed
hope you can appreciate that semi-conservative, whereby
DNA pol I doesn't need to be each of the original two strands
very good. It's only responsible is read individually to synthesize
for replicating the small region a new and complementary
encompassed by that RNA strand.
primer.
***
So,.. after this enzyme has done
its thing, you should now agree Actually, I lied. It’s not quite
with the following statement — over. Before, I finally put this Can you see that under these
that you have now doubled your whole replication thing to rest, I circumstances, the helix on
DNA. Of course, it’s still a bit think it’s also worth talking about either side of the opening will be
untidy because the strands one other enzyme, or a family of actually twisted even more. It
(especially the lagging strand) enzymes, known to scientists as would be like taking your
are still in bits and pieces. Enter topoisomerases. I like replication fork, grabbing hold of
the next and final enzyme, mentioning these enzymes, each strand, and like the
which is called the ligase. This because I think they do a helicase forcing an opening like
enzyme has only one job and wonderful job of illustrating just this:
that is to seal all of the bits and how complicated and elegant
pieces together. It fairly nature is, when confronted with
analogous to a glue job and a specific job.
essentially your picture will go
from something like this: What we’ll need to do here is
undergo a visual exercise. Let’s
say I tell you to hold two fingers
up like this:

Do you see that this will cause a

To something like this: further tightening of the coil
And let’s say that I have an along the helix?
elastic band. With this elastic
band, I will twist and coil it and This is actually very bad for the
then place it around both of your DNA molecule, as this twisting
fingers. Essentially, this will can cause a lot of structural
represent the double helix and stress. So much so, that the
will look a bit like this: DNA molecule is in very real
danger of snapping - which you
can imagine would be a very
bad thing to happen during
replication.
And (drum roll please) VIOLA!
You have doubled your DNA. ?Topoisomerases are enzymes
You have made two copies of that are designed to take care of
the same genetic code - which this problem. These enzymes
during the process of cell can actually detect these areas
division, will enable each of the If you recall, the first thing that
had to happen was for a of high structural stress, and
two new cells to receive a copy zone in on them. Not only that,
of the genome. helicase enzyme to come in and
open up that helix structure. but whilst they are at these
Let’s say that I am the helicase, areas, they will then cut both
One of the nuances that should strands in the DNA complex.
be mentioned is that if you and I come in and grab hold of
the two strands of your elastic Remarkably, they will then hold
examine the entire process, you
on to all four ends of the cut, will always be an issue of
and in a very controlled fashion, ?This is nothing short of structural stress, which is
unwind to alleviate the stress. amazing, but hopefully you can always addressed by the
Finally, they will also behave see that these enzymes play an actions of these remarkable
like ligases and stick back the important role. As the DNA is enzymes.
correct ends together again. opening up for replication, there
APPENDIX B: Hybridization/Stringency DAY 1
HYBRIDIZATION: Term defining the act of a single strand piece of nucleic acid, finding its
complement.
Very important concept, because at the end of the day, any procedure that relies
on probe or primer binding is basically a challenge of getting the hybridization to
work properly.

BUT WHAT EXACTLY IS GOING ON DURING HYBRIDIZATION?

Well you have two very negatively charged, very hydrophobic, potentially flexible, one possibly
immobilized to a charged/hydrophobic matrix, things coming together AND at the heart of it, you
want that interaction to occur because of specific H-bonding between the two molecules.

WHIC BRINGS US TO...

STRINGENCY: Need to get comfortable with this concept. High stringency means the binding
conditions are such that specificity is promoted , although too high stringency infers the
incubation conditions are too string such that nothing will interact. Low stringency means that the
binding conditions are such that binding is more easily facilitated. But, if incubation conditions are
too weak, your probe may bind to sequences that are close, but not necessarily specific.
Stringency is the difference between perfect data, dirty data or no data.

THINGS THAT EFFECT STRINGENCY: or why solutions are just so.

1. SALT CONCENTRATION. Salt will neutralize the net negative charge of nucleic acids.
More Salt = Lower Stringency. This is a condition that is especially played around with
southern/northern,microarray type of experiments. In PCR, modification of Mg ion
amounts is a reflection of this.
2. TEMPERATURE: Higher Temperature = Higher Stringency
3. DENATURATION REAGENTS: (like formamide or DMSO), essentially help ensure that
non-specific interactions do not occur because of tertiary structure formation. More =
Higher Stringency.
4. DETERGENT CONCENTRATION: alleviate hydrophobic interactions More Detergent =
Higher Stringency.
5. NUCLEIC ACID AMOUNTS: (as in the primer/probe and template). Saturating binding
conditions can lead to non-specificity. Conversely, extremely low concentrations, may
not meet the binding kinetic criteria for an interaction to occur in the first place. NOTE
that in PCR, this parameter effectively changes with each cycle (i.e. TEMPLATE amount
doubles each cycle). More stuff = Lower Stringency.
6. THE PRIMER/PROBE ITSELF: Obviously the length and specificity of the primer/probe
will impact on your stringency values. Furthermore, if labeled, the sensitivity of the type
of label will impact.

BOTTOM LINE. Is that stringency conditions will vary from situation to situation. But hopefully the
above will give you some guidelines to pursue. Follow these guidelines, make an educated
guess, look at your data and adjust the conditions accordingly…