Introduction

Bases
There are five different bases, each is denoted by a single letter as given in the parenthesis:

Adenine (A), Cytosine (C), Guanine (G), Thymine (T), and Uracil (U).
Among them,

A, C, G and T exist in DNA;

A, C, G and U exist in RNA.

A and G contain a pair of fused rings, classified as purines. C, T, and U contain only one
ring, classified as pyrimidines.

A nucleic acid chain

• Its 5' end contains a free phosphate group.
• The 3' end has a free hydroxyl group.
• In DNA or RNA, a nucleic acid chain is also called a strand.
• A DNA molecule typically contains two strands whereas most RNA molecules contain a
single strand.
• Due to the specific base pairing, DNA's two strands
are complementary to each other.
• Hence, the nucleotide sequence of one strand determines the
sequence of another strand.
• Sequence of the two strands can be written as
• 5' -ACT- 3'
• 3' -TGA- 5'

• Note that they obey the (A:T) and (C:G) pairing rule.
• If we know the sequence of one strand, we can deduce the
sequence of another strand.
• For this reason, a DNA database needs to store only the
sequence of one strand.
• By convention, the sequence in a DNA database refers to the
sequence of the 5' to 3' strand (left to right).
 "Double helix" structure
• The normal right-handed "double helix" structure of DNA, also
known as the B form
• In a DNA molecule, the two strands are not parallel, but intertwined
with each other. Each strand looks like a helix.
• The two strands form a "double helix" structure, which was first
discovered by James D. Watson and Francis Crick in 1953.
• In this structure, also known as the B form, the helix makes a turn
every 3.4 nm, and the distance between two neighboring base pairs
is 0.34 nm.
• Hence, there are about 10 pairs per turn.
• The intertwined strands make two grooves of different widths,
referred to as the major groove and the minor groove, which may
facilitate binding with specific proteins
 Famous experiments in molecular biology

• Linking genes with the synthesis of enzymes came from experiments of George
Beadle and Edward Tatum, performed in 1941 with the fungus Neurospora
crassa – one gene one protein hypothesis
• Identification of DNA as the carrier of the Genetic Information: Griffith
Experiment, Avery, Macleod, & McCarty (1944) and Hershey and case (1952)
• Chemicals can cause mutations Bruce Ames: Ames test
• Three-dimensional structure of DNA, deduced in 1953 by James Watson and
Francis Crick, has been the basis for present-day molecular biology.
• Semiconservative DNA replication was obtained in 1958 as a result of elegant
experiments, performed by Matthew Meselson and Frank Stahl, in which DNA
was labeled with isotopes that altered its density
 Bacterial Genetics
• Bacteria: E. coli - Length -2 μm
• Length of E . coli - DNA : 1.7 mm
• DNA exists as a single covalently closed double stranded circular
molecule coiled and packed within the nuclei of the cell
• Chromosome of E. coli: 4.6 million base pairs (Mb)
• In addition to chromosomal DNA, extra chromosomal circular DNA
exists in bacteria called plasmids
• Plasmids are small circular DNA molecules that can replicate
independently-without being associated with chromosomal DNA- in
bacteria
 Mutations
• Mutation refers to the change in a DNA sequence, which may
involve only a few bases or the large-scale chromosome
abnormality
• The change will be transmitted to next generation if there is
DNA replication. This cause a permanent change in the base
sequence
• Favourable mutations: confirm some advantage to the cell
• Variations necessary for natural selection during evolutions.
Mutation is the foundation of evolutionary adaptation
• Without the process of mutation, genes would not change, and
ultimately there would be no variation among organisms.
 Point mutations: just one or a few nucleotide base
pairs are affected
1. Substitutions: in which a single nucleotide is
substituted for another
• In the substitution mutation, one or more nucleotides
are substituted by the same number of different
nucleotides. In most cases, only one nucleotide is
changed.
• Based on the change in the nucleotide type, the
substitution mutation may be divided into transition
and transversion mutations.
• Based on the consequence of mutation, the substitution
mutation may be grouped into silent, missense and
nonsense mutations.
• Silent mutations: the substitution does not change the amino
acid sequence because of the redundancy of the genetic code
• Missense mutation: A change in a nucleotide sequence
resulting in a codon that specifies a different amino acid
• Nonsense mutation: a base-pair substitution changes an
amino acid codon into a stop codon

In the transition mutation, a pyrimidine (C or T) is substituted by

another pyrimidine, or a purine (A or G) is substituted by another
purine.
The transversion mutation involves the change from a
pyrimidine to a purine, or vice versa
2. Frameshift mutations: includes insertions/ deletions of
nucleotides

• In the insertion mutation, one or more nucleotides are inserted

into a sequence. If the number of inserted bases is not a
multiple of 3, it will cause frameshift

❖ Nearly all nonsense mutations result in nonfunctional proteins.

Frameshift mutations (insertions or deletions) typically result
in drastic missense and nonsense mutations.
 Recombinant DNA technology
(Generation of Recombinant DNA Molecules)
Paul Berg -1980 (Nobel prize)
• DNA cloning : DNA cloning involve separating a specific gene or DNA segment
from a large chromosome and attaching it to a carrier DNA, introducing the
recombinant DNA to bacteria and replicated the modified DNA millions of times
by increasing the cell number and creating multiple copies of cloned DNA in each
cell.
• This will help in selective amplification of the desired segment of DNA or gene
Steps in DNA cloning
1. Cutting the DNA at precise locations with sequence specific endonucleases =
restriction endonuclease = molecular scissors
2. A small DNA known as vector, capable of self replication has to be selected
3. Ligate the segment of DNA to vector covalently with the help of enzyme : DNA
Ligase
4. Transfer the rDNA to a host cell which provides the enzymatic machinery for
replication (transformation)
5. Select or identify the host cell that contain the rDNA
These methods and related tasks are collectively referred to as Recombinant DNA
technology/Genetic Engineering
• DNA/Gene/Molecular cloning is to insert a DNA fragment of interest (e.g.,
a segment of DNA) into a DNA molecule (called a vector) that is capable of
independent replication in a host cell.
• The result is a recombinant molecule or molecular clone, composed of the
DNA insert linked to vector DNA sequences.
• Large quantities of the inserted DNA can be obtained if the recombinant
molecule is allowed to replicate in an appropriate host.

Cell and Molecular Biology-Gerald Karp: John Wiley & Sons, 5th edition
 Restriction Endonucleases
• Is present in wide range of bacterial species
• Werner Arber discovered that biological function of these enzymes is to
recognize and cleave foreign DNA.
• Bacteria protect its own DNA by methylating the recognition sequences of REN
by a specific methylating enzymes
• REN and corresponding methylase in the bacteria are referred to as restriction
modification system
• There are three types of REN-Type I, II and III
• Type I and III are large multi-subunit complexes containing both endonuclease
and methylase activity
• Type I- cleaves DNA at random sites that are more than 1000bp from recognition
sequences
• Type III- cleaves DNA at about 25 bp from recognition sequence
• Both Type I and III require ATP for its function
• Type II- 1st isolated by Hamilton Smith cleave DNA at the recognition
sequence and require no ATP
• The extraordinary utility of type II enzymes was demonstrated by Daniel
Nathens who used it for first time to develop novel methods for cloning,
mapping and analyzing genes and genomes
Type II enzymes produce two types of cuts
• A :Straight Cuts (Blunt ends)
• B: Staggered Cut (Sticky ends) (Werner Arber, Hamilton Smith and
Daniel Nathens: Nobel Prize : 1978)
 Restriction Endonucleases

• Since restriction endonucleases digest DNA at specific sequences, they can

be used to cleave a DNA molecule at unique sites.
• For example, the restriction endonuclease EcoRI recognizes the
six-base-pair sequence GAATTC
 Vectors
Selection of vectors
• Plasmids: small circular extrachrosomal DNA in bacteria
• Plasmids can act as a vector
Points to be remembered while selecting a vector
1. Capacity
2. Must have origin of replication
3. Must have antibiotic resistant gene
4. Must have unique restriction sites

• Plasmids can incorporate 20 kb fragments

PBR 322, PBR 328
• Cosmids are similar to plasmids contain cos site of lamda
phage. Cos site contain sequences necessary for packaging
into its capsid
• Cosmids - can incorporate 50 kb fragments
• Pi plasmids –phage vector can incorporate upto 100 kb
fragments
• Lamda DNA- 15 to 20 kb fragments
• Bacterial artificial chromsome (BAC)- circular plasmids
–IMb/1000kb
• Yeast artificial chromsome (YAC)- 10Mb
 Cloning and expression vectors

• A cloning vector is a DNA molecule which can replicate in a

suitable host organism, and into which a fragment of foreign
DNA can be introduced.

• On the other hand expression vectors are vector that, in

addition to the properties of a cloning vector, contains
sequences that direct expression of the inserted DNA sequence
in an appropriate host organism.
• Eg pET series of expression vectors
 Transformation
• The two major methods of gene transfer by transformation are
chemical transformation and electroporation.
• The general method of transformation is the chemical
transformation in which the treatment of host cells with
calcium-chloride/heat shcok makes the cells more permeable to take
up exogenous DNA. This cell stage is called competent cells. Then,
pores are created on the cell membrane temporarily by a heat shock.
• In electroporation, cells are made competent by giving an electric
shock.
• Particle bombardment is the general method of transferring DNA
into plant cells in which DNA coated gold or tungsten are forced
into the cells physically by a gene gun.
• DH5α (Cloning), BL21 (Expression)
 Steps of bacterial transformation and selection

Specially prepared bacteria are mixed with DNA (e.g., from a ligation).
The bacteria are given a heat shock, which causes some of them to take up a plasmid.
Plasmids used in cloning contain an antibiotic resistance gene. Thus, all of the bacteria are
placed on an antibiotic plate to select for ones that took up a plasmid.
Expression vectors leads to proteins synthesis
 Transfection
• Transfection is only done in eukaryotic cells.
• Chemicals like calcium phosphate and diethylaminoehtyl
(DEAE)-dextran neutralize or even impart an overall
positive charge on DNA molecules so that it can more easily
cross the negatively charged cell membrane.
• Physical methods such as electroporation or microinjection
actually pokes holes in the cell membrane so DNA can be
introduced directly into the cell.
• Microinjection requires the use of a fine needle to deliver
nucleic acids to individual cells.
• Electroporation on the other hand uses electrical pulses to
create transient pores in the cell membrane that genetic
material can pass through.
 Applications of Recombinant DNA technology

• Recombinant DNA is widely used in biotechnology, medicine

and research.
• The most common application of recombinant DNA is in basic
research, in which the technology is important to most current
work in the biological and biomedical sciences.
• Recombinant DNA is used to identify, map and sequence
genes, and to determine their function.
• Recombinant proteins are widely used as reagents in
laboratory experiments and to generate antibody probes for
examining protein synthesis within cells and organisms.
• Many additional practical applications of recombinant DNA
are found in industry, food production, human and veterinary
medicine, agriculture, and bioengineering.
 DNA sequencing (Sanger sequencing/chain termination method)

Frederick Sanger 1980- Nobel Prize

• The most common method of DNA sequencing is based on premature termination

of DNA synthesis resulting from the inclusion of chain-terminating
dideoxynucleotides (which do not contain the deoxyribose 3‘ hydroxyl group) in
DNA polymerase reactions
• DNA synthesis is initiated at a unique site on the cloned DNA from a synthetic
primer.
• The DNA synthesis reaction includes each of the four dideoxynucleotides (A, C,
G, and T) in addition to their normal counterparts.
• Each of the four dideoxynucleotides is labelled with a different fluorescent dye, so
their incorporation into DNA can be monitored.
• Incorporation of a dideoxynucleotide stops further DNA synthesis because no 3'
hydroxyl group is available for addition of the next nucleotide.
• Thus a series of labelled DNA molecules is generated, each
terminating at the base represented by a specific fluorescent
dideoxynucleotide.
• These fragments of DNA are then separated according to size by
gel electrophoresis. As the newly synthesized DNA strands are
electrophoresed through the gel, they pass through a laser beam
that excites the fluorescent labels.
• The resulting emitted light is then detected by a photomultiplier,
and a computer collects and analyses the data.
• The size of each fragment is determined by its terminal
dideoxynudeotide, marked by a specific color fluorescence, so the
DNA sequence can be read from the order of fluorescent-labeled
fragments as they migrate through the gel.
 Maxam-Gilbert sequencing: Chemical method
• The Maxam and Gilbert method was developed in 1977 (Walter Gilbert-1980;
Nobel Prize)
• DNA extraction, DNA is denatured- heat denaturation -single-stranded DNA is
generated.
• The phosphate (5’ P) end of the DNA is removed - labeled by the radiolabeled
P32. (phosphatase removes the phosphate, the kinase adds the 32P to the 5’
end).
• 4 different chemicals are used to cleave DNA at four different positions;
hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides
while dimethyl sulfate and piperidine attack purine nucleotides.
• Hydrazine: T + C, Hydrazine NaCl: C, Dimethyl sulfate: A + G, Piperidine: G
• An equal volume of 4 different ssDNA samples is taken into 4 different tubes
each containing 4 different chemicals. The samples are incubated for
sometimes and electrophoresed in polyacrylamide gel electrophoresis. The
results of the chemicals cleavage of four different tubes are shown in the figure
below.
• Autobiography is used to visualize the separation of DNA fragments. Due to
the radiolabelled 32P end of the DNA, the DNA bands visualized through
autoradiography
 Amplification of DNA by the Polymerase Chain Reaction

• Molecular cloning allows individual DNA fragments to be

propagated in bacteria and isolated in large amounts.
• An alternative method to isolating large amounts of a single DNA
molecule is the polymerase chain reaction (PCR), which was
developed by Kary Mullis in 1988.
• The polymerase chain reaction allows the production of millions of
copies of a DNA fragment from just one starting DNA molecule
• Provided that some sequence of the DNA molecule is known, PCR
can achieve a striking amplification of DNA via reactions carried
out entirely in vitro.
• Essentially, DNA polymerase is used for repeated replication of a
defined segment of DNA.
• The number of DNA molecules increases exponentially, doubling
with each round of replication, so a substantial quantity of DNA
can be obtained from a small number of initial template copies
• The multiple cycles of heating and cooling involved in PCR are
performed by programmable heating blocks called thermocyclers.
• The DNA polymerases used in these reactions are heat-stable
enzymes from bacteria such as Thermus aquaticus (Taq DNA
Polymerase) from Thermus aquaticus, which lives in hot springs at
temperatures of about 75°C.
• These polymerases are stable even at the high temperatures used to
separate the strands of double-stranded DNA, so PCR amplification
can be performed rapidly and automatically.
• For example, a single DNA molecule amplified through 30 cycles of
replication would theoretically yield 2 raised to30 (approximately 1
billion) progeny molecules
 Primers
• Primers are single-stranded DNA molecules usually synthesized commercially, i.e.,
polynucleotides of variable sizes.
• These short polynucleotide DNA (18 to 25 bp) strands have a free 3′ hydroxyl
group, also called as 3′ end.
• The free 3′ hydroxyl group on the primer is needed by the DNA polymerase to add
new nucleotides during the polymerization process, thereby synthesizing a new
complementary strand
• The binding of DNA primer to the target requires the separation of two
complementary DNA strands (Denaturation) which is generally achieved by heating
process
• To perform PCR, two primers are needed to enhance both the strands of the
template: a primer for one strand (or sense strand), called the “forward primer,”
which is the beginning of the template, and another primer for the complementary
strand (or the antisense strand) called the “reverse primer.” Thus, both the primers
bind to 5′ ends of the sense and antisense strand
 Application
Diagnosis of infections
Diagnosis of genetic defects
Diagnosis and prognosis of cancers
Phylogenetics
Archeology
Recombinant DNA technology
Metagenomics
Site-directed mutagenesis
Personalized medicine
Forensics sciences
DNA profiling
Gene expression profiling
Identifying medicinal plants
Detecting GMO
Meat traceability
 Hybridization

Nucleic Acid Hybridization

• The key to detection of specific nucleic acid sequences is base
pairing between complementary strands of RNA or DNA.
• At high temperatures (e.g., 90 to 100°C) the complementary
strands of DNA separate (denature), yielding single-stranded
molecules.
• If such denatured DNA strands are then incubated under
appropriate conditions (e.g., 65°), they will renature to form
double-stranded molecules as dictated by complementary base
pairing-a process called nucleic acid hybridization.
• Nucleic acid hybrids can be formed between two strands of DNA,
two strands of RNA, or one strand of DNA and one of RNA.
• Probe :Radioactivity/Flourscently labeled DNA/RNA used in
hybridization experiment to detect its complementary region in the
genome
Detection of DNA by nucleic acid hybridization

• A specific sequence can be detected in total cell DNA by hybridization with a

labelled DNA probe, containing radioactive nucleotides or modified
nucleotides that can be detected by fluorescence or chemiluminescence.

• The DNA is denatured by heating to 95°C, yielding single-stranded

molecules.

• The labelled probe is then added and the temperature is lowered to 65°C,
allowing complementary DNA strands to renature by pairing with each other.

• The probe hybridizes to complementary sequences in cell DNA, which can

then be detected by incorporation of the labelled probe into double-stranded
molecules.
 Southern blotting

• A technique developed by E. M. Southern is widely used for

detection of specific genes in cellular DNA .
• The DNA to be analyzed is digested with a restriction
endonuclease, and the digested DNA fragments are separated
by gel electrophoresis.
• The gel is then overlaid with a nitrocellulose filter or nylon
membrane to which the DNA fragments are transferred
(blotted) to yield a replica of the gel.
• The filter is then incubated with a labeled probe, which
hybridizes to the DNA fragments that contain the
complementary sequence, allowing visualization of these
specific fragments of cell DNA.
 Northern blotting

• Northern blotting is a variation of the Southern

blotting technique (hence its name) that is used for
detection of RNA instead of DNA.
• ln this method, total cellular RNAs are extracted and
fractionated according to size by gel electrophoresis.
• As in Southern blotting, the RNAs are transferred to
a filter and detected by hybridization with a cloned
probe.
• Northern blotting is frequently used in studies of
gene expression-for example, to determine whether
specific mRNAs are present in different types of cells
 Western blotting
• Western blotting is another variation of Southern blotting.
• Proteins in cell extracts are first separated according to size by gel
electrophoresis.
• Because proteins have different shapes and charges, however, this
process requires a modification of the methods used for
electrophoresis of nucleic acids.
• Proteins are separated by a method known as SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) in which they are dissolved in a
solution containing the negatively charged detergent sodium
dodecyl sulfate (SDS).
• Each protein binds many detergent molecules, which denature the
protein and give the protein an overall negative charge.
• Under these conditions, all proteins migrate toward the positive
electrode-their rates of migration determined (like those of nucleic
acids) only by size.
• Following electrophoresis, the proteins are transferred to a filter,
which is then allowed to react with antibodies against the protein of
interest.
After SDS-PAGE, a membrane is placed on the gel, to which the separated proteins in the gel are
electrophoretically transferred. The membrane with transferred proteins is then probed with a
primary antibody (an antibody specific for the target protein), washed, and reacted with a secondary
antibody labeled with an enzyme, such as horseradish peroxidase (HRP). The bound enzyme
activity is used to detect the target protein and visualized by a chemiluminescent or chromogenic
method.
The membrane with transferred proteins is blocked to prevent non-specific binding of
antibodies to the membrane.
 Genomic Library
• Fragment of DNA arranged in a particular way (Bacteriophages, BAC,
YAC are used)
Procedure
1. Isolation of Genomic Library
2. Making fragments with RENs
3. Separation of Fragments
• Upto 20 kb (Agarose gel electrophoresis)
• Large fragments (Pused Field Gel Electrophoresis (PFGE)
• Gels stained with Ethidium bromide
• Molecular size markers are used to cut fragments of desired size
• Agarose gel cuts will be digested with enzyme agarase
4 Select Vector of choice
• Digest Vector with same REN
5. Fragments of suitable size will be incorporated to vector
• Fragments ligated to vector using DNA ligase
6. Bacterial transformation: transfer of recombinant plasmid into bacteria
7. Transformed bacteria is cultured
8. Agar plate containing suitable antibiotic are used
• Each bacteria with unique fragment cloned are preserved in 96
or 394 well plates
• Whenever a investigator want to isolate a particular sequence ,
bacteria are grown again and screened for necessary genetic
elements
• Use of randomly cleaved DNA has an advantage in
construction of library because it generate overlapping
fragments that can be used in the analysis of regions of the
chromosome extending out in both directions from a particular
sequence , a technique known as chromosomal walking
 Bioinformatics, Gene banks
• Traditionally, molecular biologists have studied one or a few genes or
proteins at a time. This has been changed by the genome sequencing
projects, which introduced new large-scale experimental approaches in
which vast amounts of data were generated.
• Handling the enormous amounts of data generated by whole genome
sequencing required sophisticated computational analysis and spawned the
new field of bioinformatics, which lies at the interface between biology
and computer science and is focused on developing the computational
methods needed to analyse and extract useful biological information from
the sequence of billions of bases of DNA.
• Eg Basic Local Alignment Search Tool (BLAST) is a sequence similarity
search program that can be used to quickly search a sequence database for
matches to a query sequence
• Gene banks are a type of biorepository which preserve genetic material
 Gene Banks

• Gene Banks are a type of bio repository which preserve genetic material.
• A collection of seed plants, tissue cultures etc. from potentially useful
species , especially species containing genes of significance to the breeding
of crops.
• The database of the largest gene banks in the world can be queried via a
common website, Genesys.
National Centre for Veterinary Type The Microbial Type Culture Collection and
Cultures (NCVTC) - established in June Gene Bank (MTCC), a national facility
2005 serves as a National repository established in 1986 is funded jointly by the
responsible for conservation of Department of Biotechnology (DBT) and the
microbes wherein microbial Council of Scientific and Industrial Research
biodiversity of animal origin is being (CSIR), Government of India. The MTCC,
conserved so that it can be utilized housed at the Institute of Microbial
effectively Technology (IMTECH), Chandigarh
 Intellectual property (IP)

• Intellectual property is the foundation of innovations. The

science of animal biotechnology has progressed vastly
during past three decades.
• The term intellectual property (IP) is generic legal term that
describes various types of intangible assets and laws which
protect the innovations and applications of thoughts, ideas, and
information of profitable value.
• In broader sense, IP protection is about the laws related to
patents, copyrights, trademarks, trade secrets, and other similar
rights
Types of IPs in Animal Biotechnology

• Patenting Genetically Modified Organisms

o Patents cover various technologies used to modify mice or rat
genome and the stem cells
• IP Issues in In Silico Biology
o Bioinformatics and in silico methods are indispensable tools to
analyze gigantic “ omics” sequence data. The bioinformatics
programs are used to predict hypothetical gene, proteins, and
metabolic pathways from the sequence data of microorganism
• Therapeutics and IP
o Vaccines, monoclonal antibodies, and antibiotics provide
protection against infectious diseases. As there are many forms of
vaccines, and components of vaccines, delivery systems, and
distribution networks, a variety of IP protections are applicable to
vaccines
 Regulatory bodies
• The Convention on Biological Diversity (CBD) Article 19 deals with the
safe transfer, handling and use of any genetically modified organism
resulting from Biotechnology.
• Bio-safely is a critical element in the CBD which addresses the
Conservation and sustainable use of Biodiversity in all its aspects i.e.,
Biological Diversity including the genetic resources associated with it.
• India has a well-defined regulatory mechanism for development and
evaluation of GMOs and the products thereof. The Department of
Biotechnology (DBT) and the Ministry of Environment & Forests (MoEF)
are the two apex regulatory bodies.
 Quantification of Nucleic Acids
• Measuring the intensity of absorbance of the DNA solution at
wavelengths 260 nm and 280 nm is used as a measure of DNA
purity.
• DNA absorbs UV light at 260 and 280 nanometres, and
aromatic proteins absorb UV light at 280 nm; a pure sample of
DNA has a ratio of 1.8 at 260/280 and is relatively free from
protein contamination.
• A DNA preparation that is contaminated with protein will have
a 260/280 ratio lower than 1.8 where as ratio higher than 1.8
indicates RNA contamination
 Electrophoresis

• Migration of a charged particle under the influence of electric

current. A method of separating large molecules (such as DNA
fragments or Proteins ) from a mixture of similar molecules.
• An electric current is passed through a medium containing the
mixture, and each kind of molecule travels through the medium at
a different rate, depending on its electrical charge and size.
• Separation is based on these differences.
❖ Agarose and acrylamide gels are the media commonly used for
electrophoresis of nucleic acids and proteins.
• Agarose is used for nucleic acids while polyacrylamide is used to
separate proteins/ small oligonucleotides
.
 Agarose Gel Electrophoresis
• DNA is negatively charged
• When placed in an electrical field, DNA will migrate toward the positive pole
(anode).
• An agarose gel is used to slow the movement of DNA and separate by size.

• Polymerized agarose is porous, allowing for the movement of DNA

• Strength of the electrical field, buffer, density of agarose gel etc can influenze
migration

Size of the DNA!

o Small DNA move faster than large DNA and gel so electrophoresis separates DNA
according to size

• Within an agarose gel, linear DNA migrate inversely proportional to the log10 of
their molecular weight
 Visualizing the DNA fragments

• When a gel is stained with a DNA-binding dye and placed under UV

light, the DNA fragments will glow, allowing us to see the DNA
present at different locations along the length of the gel.

• DNA is visualized by including in the gel an intercalating dye,

ethidium bromide. DNA fragments take up the dye as they migrate
through the gel. Illumination with ultraviolet light causes the
intercalated dye to fluoresce with a pale pink colour.
 Polyacrylamide gel electrophoresis
(SDS-PAGE)
• When proteins are separated by electrophoresis through a gel matrix,
smaller proteins migrate faster due to less resistance from the gel matrix.
Other influences on the rate of migration through the gel matrix include the
structure and charge of the proteins.

• In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as

sodium lauryl sulfate) and polyacrylamide gel largely eliminates the
influence of the structure and charge, and proteins are separated solely
based on polypeptide chain length.

• SDS is a detergent with a strong protein-denaturing effect and binds to the

protein backbone at a constant molar ratio. In the presence of SDS and a
reducing agent that cleaves disulfide bonds critical for proper folding,
proteins unfold into linear chains with negative charge proportional to the
polypeptide chain length
• Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix
suitable for the separation of proteins of typical size. The strength of the gel
allows easy handling. Polyacrylamide gel electrophoresis of SDS-treated
proteins allows researchers to separate proteins based on their length in an
easy, inexpensive, and relatively accurate manner.
 Polyacrylamide Gel Electrophoresis
• Monomeric acrylamide (which is neurotoxic) is polymerized in the
presence of free radicals to form polyacrylamide.
• The free radicals are provided by ammonium persulphate and
stabilized by TEMED (N'N'N'N'-tetramethylethylene-diamine).
• The chains of polyacrylamide are cross-linked by the addition of
methylenebisacrylamide (bis) to form a gel whose porosity is determined by the
length of chains and the degree of crosslinking.
• Polyacrylamide gels are poured between two glass plates held apart
by spacers of 0.4 - 1.0 mm and sealed with tape.
• Most of the acrylamide solution is shielded from oxygen so that inhibition of
polymerization is confined to the very top portion of the gel.
• The length of the gel can vary between 10 cm and 1m depending on
the separation required.
.
• Polymerization initiated Ammonium Persulphate (1.5%,W/v)
provides free radicals and TEMED (N,N,N,N-tetra-methylene
ethylene-diamine) acts as activator of reaction.
• Concentration of acrylamide and Bisacrylamide solution is
increased, the pore size of the gel is decreased.
• Advantage of acrylamide gel systems is that initial concentrations
of acrylamide and BIS control the hardness and degree of
crosslinking of gel.
• Generally resolving gels varying between 10-15% and stacking
gels of 5% are used.
 Protocol
1. Grow culture in 5 ml broth, pellet cells (~3000 g, 10 min), discard supernatant.
2. Add 400 µl TE and 100 µl NaCl (5 M) to pellet, resuspend cells by vortexing.
3. Add 50 µl CTAB, vortex, incubate 60°C for 20 min, occasionally mixing by
inversion of tube.
4. Add 500 µl chloroform, vortex and mix thoroughly, incubate on ice 30 min.
5. Spin at ~10,000 g in microfuge (cold if possible) for 10 min.
6. Slowly pipette out and retain supernatant – avoid interface material and lower
phase.
7. Add 500 µl phenol:chloroform, vortex until milky solution obtained (~20 sec),
spin at top speed in microfuge for 5 min, retain supernatant.
8. Add 500 µl chloroform, vortex, spin 5 min, retain supernatant.
9. Discard all waste phenol and chloroform into chemical waste bottle in fume hood.
10. Add 1/10 volume Na-acetate (50 µl) and 2 vol. ice-cold ethanol (1000 µl) to
precipitate DNA – incubate at -20°C for at least 1 hour.
11. Spin at top speed in microfuge (cold if possible) 10 min, drain off ethanol, retain
pellet.
12. Add 500 µl 70% ethanol, resuspend pellet by flicking,
allow to sit for ~5 min at room temp, then spin and drain again.
13. Invert tube on paper towel with lid open for ~5 min to drain
last bits of 70% ethanol, then transfer to 60°C incubator (OK if
tube is lying on its side, but lid open) for 10 min to dry residual
ethanol.
14. Redissolve pellet in 100 µl EB. Heating at 50-60°C (up to
an hour, intermittent mixing) and/or addition of more EB may
be required to dissolve all the DNA.
15. Store final DNA preparation at -20°C. If you will be using
it a lot, consider making multiple smaller aliquots, since
repeated freeze/thawing will deteriorate the quality
 Genomic DNA Prep Solutions

• TE: 10 mM Tris-HCl, 1 mM EDTA. pH 8.0 Autoclaved.

• NaCl: 5 M NaCl. Autoclaved.
• CTAB: 10% w/v CTAB. Autoclaved. May need heating (~50°C) to
dissolve before each use.
• Chloroform: Mixture of chloroform and isoamyl alcohol (24:1
ratio) equilibrated with a layer of TE buffer (pH 8) on top. Not
sterilized.
• Phenol:chloroform: Mixture of phenol, chloroform, isoamyl
alcohol (25:24:1 ratio) equilibrated with a layer of TE buffer (pH
8) on top. Not sterilized.
• Na-acetate: 3M sodium acetate in water, sterilise by autoclaving
(don’t need to adjust pH)
• EB (Elution buffer): 5 mM Tris-HCl. pH 8.0 Autoclaved.
 Plasmid Isolation Using Alkaline Lysis

• The purification protocol therefore involves a differential

precipitation step, in which the long strands of E. coli DNA,
entangled in the remnants of lysed cells, are preferentially
removed.
• Because each of the complementary strands of plasmid DNA is
a covalently closed circle, the strands cannot be separated
(without breaking one of them) by conditions such as exposure
to mild alkali (up to pH 12.5), which break most of the
hydrogen bonds of DNA.
• Closed circular molecules regain their native configuration
when returned to neutral pH. E. coli remains in the denatured
state.
1. Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage.
2. Discard the supernatant.
3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent
metals (primarily magnesium and calcium). Removal of these cations destabilizes
the cell membrane. It also inhibits DNases. Glucose should also be added to
maintain osmolarity and prevent the buffer from bursting the cells.
4. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline
solution gave rise to the name of this technique. Mix this by gentle inversion and
incubate on ice for five minutes (but no longer, or your DNA will be irreversibly
denatured). Three things happen during this stage:
a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate)
is a detergent.
b. NaOH loosens the cell walls and releases the plasmid DNA and sheared
cellular DNA.
c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands
are separated. Plasmid DNA is circular and remains topologically constrained.
.
5. Renature the plasmid DNA and get rid of the garbage. Add potassium
acetate (KAc), which does three things:
a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured
as single stranded DNA (ssDNA).
b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high
salt.
c. Adding acetate to the SDS forms KDS (Potassium dodecyl sulfate., which is
insoluble. This will
allow for the easy removal of the SDS from your plasmid DNA.
6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or
isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium
chloride or sodium acetate) and spin this down. DNA is negatively charged, so
adding a salt masks the charges and allows DNA to precipitate. This will place
your DNA in the pellet.
7. Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for
about 10 minutes to allow the EtOH to evaporate.
8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus
RNases to cleave any remaining RNA. Your DNA is now back in solution.
 Plasmid Isolation Using Alkaline Lysis
1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial colony.
The tube was incubated at 37 ˚C overnight with vigorous shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet up and down
or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS (solution II) to the suspension. Mix
thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion. Do
not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated gentle
inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to
evaporate.
13. Add ddH2O or TE to dissolve the pellet. After addition of 2ul RNase A (10mg/ml), the mixture
was incubated for 20 minutes at room temperature to remove RNA.
• Solution I (Buffer I): 50 mM glucose, 10 mM EDTA, 25 mM
Tris, pH 8.0. Store at 0˚C a. 10ml 500mM Glucose b. 2ml
500mM EDTA pH 8.0 c. 2.5ml 1M Tris pH 8.0 d. 85.5ml H2O
e. Autoclave and store at 4°C
• Solution II (Buffer II): Freshly prepared 0.2 N NaOH, 1%
SDS. Store at room temperature (RT) Isopropanol: Stored at
-20 0˚C
• Solution III (Buffer III): 3M KOAc, pH 6.0 a. 60ml 5M
potassium acetate (49.07g potassium acetate in 100ml H2O) b.
11.5ml glacial acetate c. 28.5ml H2O
 Plasmid profiling
• Plasmids are circular deoxyribonucleic acid molecules that
exist in bacteria, usually independent of the chromosome.
Plasmids can also serve as markers of various bacterial strains
when a typing system referred to as plasmid profiling, or
plasmid fingerprinting is used.