Chapter 3

Recombinant DNA Technology and Genomics

Chapter Contents
• 3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• 3.2 What Makes a Good Vector?
• 3.3 How Do You Identify and Clone a Gene of
Interest?
• 3.4 Laboratory Techniques and Applications of
Recombinant DNA Technology
• 3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
Gene cloning
 • 1970s: Gene cloning became a reality

• Clone – a molecule, cell, or organism that was produced from

another single entity

• Made possible by the discovery of:

• Restriction Enzymes – DNA cutting enzymes (molecular scissors)

• Plasmid DNA Vectors – circular form of self-replicating DNA

• Can be manipulated to carry and clone other pieces of DNA

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 What do we need to do cloning?

Plasmid

Restriction Enzymes
Restriction Enzymes

Scissor that cut DNA

 Restriction Enzymes
6 bp cutters

• Primarily found in bacteria

• Cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotides
in a DNA strand

• Bind to, recognize, and cut DNA within specific sequences of bases called a
restriction site

• There are 4 or 6 bp cutters because they recognize restriction sites with a

sequence of 4 or 6 nucleotides

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 a. Some cut DNA to create DNA fragments with overhanging
single stranded ends called "sticky" or "cohesive" ends

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b. Some cut DNA to generate fragments
with double-stranded ends called
"blunt" ends
 • Why don't restriction enzymes digest bacteria DNA?
some of the nucleotides in their DNA contain methyl groups that
block restriction enzymes from digestion
© 2013 Pearson Education, Inc. Ch3 Figure 1 (textbook)
Ch3 Table 1(textbook)
 • Advantage of enzymes that produce sticky ends

• Preferred for cloning because DNA fragments with sticky

ends can be easily joined together because they base pair
with each other by forming weak hydrogen bonds

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 What do we need to do cloning?

Plasmid

Restriction Enzymes
Plasmid DNA
 Plasmid DNA
• Plasmid DNA – small circular pieces of DNA found
primarily in bacteria
• Are considered extrachromosomal DNA because they
are in the cytoplasm in addition to the bacteria
chromosome
• Are small approximately 1 to 4 kb
• Can replicate independently of chromosome
• Can be used as vectors – pieces of DNA that can
accept, carry, and replicate other pieces of DNA
 A Typical Plasmid for cloning must have:
To replicate inside
bacteria cells
Ori

To select
transformed
bacteria cells To clone
gene/DNA in
this site
How do we perform a cloning experiments?

1- Restriction Enzyme cut and

ligation (cloning)

2- Transformation

3- Selection
 Step 1: Restriction Enzyme cut and ligation
between vector and insert DNA

Cut BOTH with

same
Restriction
Enzymes
 Creating recombinant DNA
EcoR1 EcoR1

Cut BOTH
with same
Restriction
Enzymes Ligate

Recombinant DNA
 Creating recombinant DNA
HindII EcoR1
Cut BOTH
with same
Restriction
Ligate
Enzymes

Recombinant DNA
 Recombinant DNA

A term used to describe the combination

of two DNA strands that are constructed
artificially
Ch2 Figure 14 (textbook)
Step 2:Transformation of Bacterial Cells
 Transformation of Bacterial Cells

• very inefficient process

• A process for inserting foreign DNA into bacteria

• Plasmid DNA enters bacterial cells and is replicated and

express their genes

• Two different methods:

• 1) By calcium chloride +heat shock
• 2) By Electroporation (most commonly used)
 Transformation of Bacterial Cells

By calcium chloride +heat shock

• Treat bacterial cells with
calcium chloride

• Add plasmid DNA to cells

chilled on ice

• Heat the cell and DNA

mixture

• Plasmid DNA enters bacterial

cells and is replicated and
express their genes
 Transformation of Bacterial Cells

Electroporation

Apply brief pulse of high voltage electricity to create tiny

holes in the bacteria cell wall that allow the DNA to enter
Step 3: Selection of transformed bacteria cells
 Selection of transformed bacteria cells

• Selection is a process designed to facilitate the identification

of recombinant bacteria while preventing the growth of non-
transformed bacteria and bacteria that contain plasmid
without foreign DNA

- We can use one of these methods:

1-Antibiotic selection
2- Blue-white selection
 Remember- A Typical Plasmid for cloning must have:

Ori

Antibiotic
Selection

To select
transformed
bacteria cells
Exa
mple:
Ampicillin
Kanamycin
 Selection
1. Antibiotic selection:

plate transformed cells

on plates containing
different antibiotics to
identify recombinant
bacteria and non-
transformed bacteria
 Selection
2. Blue-white selection

• DNA is cloned into the restriction site in the lacZ gene

• When it is interrupted by an inserted gene, the lacZ gene
cannot produce functional Beta gal Enzyme
• When Xgal (artificial lactose) is added to the plate, if
functional lacZ is present = blue colony
• Non-functional lacZ = white colony = clone = genetically
identical bacterial cells each containing copies of recomb.
plasmid
2. Blue-white selection
 Ch3 Figure 3 (textbook)

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 • Assume you used a plasmid that contains the lacz
gene in the restriction enzyme site. The plasmid has
an antibiotic resistance gene. Following
transformation, you grow up the cells on an agar plate
containing the antibiotic. Here are your results.

1
2

3 4

5 6

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 Introduction to human gene cloning

• First human protein expressed via recombinant

techniques was insulin and next was growth hormone

• Clone human insulin DNA sequence into a plasmid and

the bacteria cells were then used to synthesize the
protein product of the cloned gene

• Can generate lots of pure protein via this technique

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 Chapter Contents
• 3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• 3.2 What Makes a Good Vector?
• 3.3 How Do You Identify and Clone a Gene of Interest?
• 3.4 Laboratory Techniques and Applications of
Recombinant DNA Technology
• 3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology

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3.2 What Makes a Good Vector?

What is a cloning vector?

A cloning vector is a small piece of DNA, taken from

a virus, a plasmid, or the cell of a higher organism,
that can be stably maintained in an organism, and
into which a foreign DNA fragment can be inserted
for cloning purposes.
 What do we need to do cloning?
Vectors
Plasmid

Restriction Enzymes
 Practical Features of DNA Cloning Vectors
To replicate inside
bacteria cells
Ori

Selectable marker genes Also called

MCS: Multiple
Cloning Site

To select
transformed
bacteria cells To clone
gene/DNA in
this site
 Practical Features of DNA Cloning Vectors

• Size – small enough to be separated from chromosomal DNA

of host plasmid
• Origin of replication (ori) – site for DNA replication that allow
plasmids to replicate independently from host chromosome
• Copy number: number of plasmids in the cell (normally small but
plasmids have high copy numbers)
• Multiple cloning site (MCS) – recognition sites for several
restriction enzymes in which insert is cloned into
• Selectable marker genes – allow to select for transformed
colonies
• RNA polymerase promoter sequences – used for transcription
in vitro and in vivo
• DNA sequencing primers

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 Is Plasmid Vectors the ONLY type of vectors we can
use?

Plasmid
Vectors

Answer: No, There are many other types of vectors we

can use for cloning. Depends on the size of the inserted
DNA you want to clone. Plasmid vectors however is the
most widely use for small insert DNA size
Example of very common plasmid vector : pUC19
 Types of vectors we can use for cloning experiments

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 Types of Vectors

• Bacterial plasmid vectors – can clone inserts that are

smaller than 7 kb
• Bacteriophage vectors
• Cosmid vectors
• Bacterial Artificial Chromosomes (BAC)
• Yeast Artificial Chromosomes (YAC)
• Ti vectors

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Bacteriophage (λ) vectors
–clone up to 25 kb insert DNA
–λ genome is linear and 49 kb
Using Bacteriophage (λ) vector for cloning
 Cosmid vectors: COS sites +Plasmid
A cosmid is a plasmid that carries bacteriophage lambda's cos gene
• COS ends of λ DNA ( for cloning)- circular
• clone fragments between 20–40 kb

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Using cosmids
for cloning
 Ti vector: Ti (Tumor inducing) vector
Plant ONLY

• Naturally occurring plasmids isolated from the bacterium

(Agrobacterium) that is a soil plant pathogen causing
disease in plants

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 Crown Gall
disease
Caused by

Agobacterium

Which has Ti
plasmid

Ti: Tumor
inducing
Plasmid
 In Nature
• When the bacteria infects plant cells, the T DNA from the Ti
plasmid inserts into the host chromosome
• T DNA codes for auxin hormone that weakens plant cell wall
and infected plants divided and enlarge to form a tumor (gall)
 In biotechnology labs

• Scientists use Ti vectors to deliver genes to plants by removing

toxic gene for auxin and inserting any other gene of interest

Ti plasmid vector
ONLY for plant
transformation
experiments
3.3 How Do You Identify and Clone a Gene of
Interest?
What is a library?

a collection of sources of information and

similar resources including books
 DNA Libraries
Collections of cloned DNA
fragments from a
particular organism
contained within bacteria
or viruses as the host

Screen the library to pick out different genes of

interest
 Two Types of Libraries

1) Genomic DNA libraries

Contains all possible fragments of DNA from a given cell or
organism
(coding: exons + non-coding: introns)

2) Complementary DNA libraries (cDNA libraries)

Carries only expressed genes
( Exons ONLY)

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 What is the different between genomic library and cDNA library?

Think about
the source of
Genomic starting
library material for
starting point both

cDNA library
starting point
 Remember we discussed in Lecture #5 mRNA processing
mRNA Processing in the nucleus Exon: coding part for the
gene
Primary Intron: non-coding part
transcript (needs to be removed)

3)Addition of a 5' cap 2) 3' PolyA tail

1) RNA splicing

mRNA

NOW : this mRNA is ready to move to

cytoplasm to start Translation process

cDNA library starting point is mRNA

What is the different between genomic library and cDNA library?
Genomic
library
starting point

cDNA library
starting
point
Assume you want to make lots of human insulin using a bacteria
expression vector.
 why using human insulin genomic DNA
for this cloning project would not be
advantageous.
Eukaryotic DNA is processed to splice out introns; add 5' cap
and 3' poly A tail. The introns will not be spliced out since
bacteria does not have an ability to process RNA. Thus, the RNA
transcribed by the bacteria RNA Pol will include the introns if
the genomic DNA is used and then it will code for the wrong
protein.
 Genomic Libraries
1) Chromosomal DNA from the tissue of interest is isolated
and digested with a restriction enzyme which produces many
fragments that include the entire genome

2) Vector is digested with same enzyme

3) DNA ligase is used to ligate genomic DNA fragments and

vector DNA

4) Recombinant vectors are used to transform bacteria and

theoretically each bacteria will contain a recombinant
plasmid

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 Genomic Libraries
vector

cleave vector with same

restriction enzyme

Bacteria Transformation
followed by selection of
transformansts
 • Disadvantages of genomic libraries

• Introns are cloned in addition to exons;

• Majority of genomic DNA is introns in eukaryotes so majority of the
library will contain non-coding pieces of DNA

• Many organisms have very large genome, so

searching for gene of interest is difficult

• Time consuming!

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 cDNA Libraries

1) mRNA from tissue of interest is isolated

2) Need to make double stranded DNA from mRNA: How? Steps

to follow (a-c):
a. enzyme reverse transcriptase catalyzes synthesis of
complementary single stranded DNA from mRNA
***Called complementary DNA (cDNA) because it is an
exact copy of the mRNA
b. mRNA is degraded either with an enzyme or alklaline solution
c. DNA Polymerase is used to synthesize second strand of DNA to create
double stranded cDNA

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Steps for making cDNA from mRNA

Step 1

Step 2

Step 3
 cDNA Libraries

3) Add Short linker double stranded DNA sequences to

the ends of the cDNA
*** LINKER contain restriction enzyme recognition sites

4) Cut cDNA with restriction enzyme, cut vector with

same enzyme

5) Ligate fragments to create recombinant vectors

6) Transform bacteria with recombinant vectors

Then, Add Linkers to the
double stranded DNA before
proceeding to 4

By cleaving DNA and vectors with

same Restrictione Enzyme

Follow with bacteria

transformation and
selection of
transformants
 Comparison of a Human Genomic library (left) and a cDNA library (right)

Ch3 Figure 4 (textbook)

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 Advantages of cDNA libraries over genomic libraries
• Collection of actively expressed genes in the cells or tissues from which
the mRNA was isolated

• Introns are NOT cloned

• Can be created and screened to isolate genes that are primarily

expressed only under certain conditions in a tissue

• Disadvantage of cDNA libraries

• Can be difficult to make the cDNA library if a source tissue with an
abundant amount of mRNA for the gene is not available

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 Colony Hybridization

Library screening with a DNA probe to

identify a cloned gene of interest

IS
 What is a probe?

DNA fragment that is complementary to the gene of interest

Loris
The probe with can be tagged with radioactive nucleotide or fluorescent
dye that can be used to catalyze light releasing reactions. Why?

Answer: To track the probe to determine where it binds

Tracking the probe by radioactive nucleotide or fluorescent dye
• Probe binds by hydrogen bonding to complementary
sequences on the filter = hybridization
Anywhere probe
bounds to the target
sequence,
radioactivity from
the radioactive
probe or released
light (fluorescence)
from non-radioactive
probes exposes
silver grains in the
film
 Type of probe used depends on what is already known
about the gene of interest

Example: use mouse

or rat probe to
screen a human
library because many
genes between these
species are similar
 Library screening to identify the gene of interest

• Colony hybridization
Start: Bacterial colonies containing recombinant DNA are
grown on an agar plate
1) Nylon or nitrocellulose filter is placed over the plate and
some of the bacterial colonies stick to the filter at the exact
location they were on the plate
2) Nitrocellulose filter is removed
3) Treat filter with alkaline solution to lyse the cells and
denature the DNA
• Denatured DNA binds to filter as single-stranded DNA
4) Filter is incubated with a probe that is tagged with a
radioactive nucleotide or fluorescent dye

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Colony Hybridization ieplate 1colonies Ii
UKOUM (1)
Start

(2)

heagt (6)

(3) (5)

Add (4)
the
 • Colony Hybridization
• Filter is washed to remove excess unbound probe
• Filter is exposed to film – autoradiography
• Anywhere probe has bound to the filter, radioactivity from the
radioactive probe or released light (fluorescence) from non-
radioactive probes exposes silver grains in the film
• Depending on the abundance of the gene of interest there might be few
colonies or plaques on the filter that hybridize to the probe
t
• Film is developed to create a permanent record of the colony
hybridization
5) Film is then compared to the original agar plate to identify
which colonies contained recombinant plasmid with the
gene of interest
6) Positive colonies are picked for further analysis

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 Colony Hybridization

Ch3 Figure 5 (textbook)

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 DN
Atlus
Library screening
64
rarely results in the
cloning of the full-length gene
ol
• Usually get small pieces of the gene; the pieces are
sequenced and scientists look for overlapping sequences

• Look for start and stop codons to know when the full
length of the gene is obtained

d
o
Library screening rarely results in the
cloning of the full-length gene
Readthegene
 Chapter Contents
• 3.1 Introduction to Recombinant DNA
Technology and DNA Cloning

4s • 3.2 What Makes a Good Vector?

• 3.3 How Do You Identify and Clone a Gene of Interest?
• 3.4 Laboratory Techniques and Applications of
Recombinant DNA Technology
• 3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology

© 2013 Pearson Education, Inc.

 Fluorescent In-Situ Hybridization (FISH)

• Technique for Detecting Chromosome

Location and Copy Number
• Identify which chromosome contains a gene of
interest

• Use for karyotype analysis

• Relies on using fluorescent DNA or RNA

probe for gene of interest
 Fluorescent In-Situ Hybridization (FISH)

Chromosome
number
Femell
Fluorescent In-Situ Hybridization (FISH)
FISH used to analyze genetic disorders

Chromosenfs
FISH used to determine which cells in a particular
organ are expressing the particular mRNA
FISH uses fluorescent DNA or RNA probe for gene of interest

The probe has a

complementary
sequence to the
gene of interest

Different
Fluorescent
dyes can be
added to the
probe
 • Procedure:

• Chromosomes are isolated from cells and spread out on glass

microscope slide

• DNA or RNA probe for gene of interest is labeled with fluorescent

nucleotides and incubated with slides

• Probe will hybridize with complementary sequences on chromosomes

on slide

• Slide is washed and exposed to fluorescent light

• Wherever probe has bound to the chromosome, it is illuminated to

indicate the presence of the probe binding

• Do karyotype to determine which chromosome shows fluorescence

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Fluorescent In-Situ Hybridization (FISH) procedure

G
Fluorescent In-
Situ
Hybridization
(FISH)

Ch3 Figure 14 (textbook)

 Polymerase Chain Reaction (PCR)

Technique for making copies, or amplifying, a

specific sequence of DNA in a short period of
time
Polymerase Chain
Reaction (PCR)
 Components of PCR
Target DNA to be amplified is added to a tube,
mixed with the following:

1- nucleotides (dATP,
dCTP, dGTP, dTTP)
2- buffer
3- Taq DNA polymerase
4- Paired set of Forward
and Reverse Primers
Primers are complementary to nucleotides flanking
opposite ends of target DNA
Primers are complementary to nucleotides flanking
opposite ends of target DNA
Reaction tube is placed in an instrument called
a thermocycler
 PCR Process

• Thermocycler will take DNA through a series of reactions called a

PCR cycle
• Each cycle consists of three stages
1. Denaturation – heat to 94 °C to 96 °C
2. Annealing (hybridization) – in which primers H bond with
complementary bases at the opposite ends of target
sequence at 55 °C to 65 °C
3. Extension (elongation) – DNA Pol copies target DNA at 70 to
75 °C
• At the end of one cycle, the amount of DNA has doubled
• Cycles are repeated 20–35 times
 PCR Process

This one cycle, this process is repeated over for every cycle for a total of 20-35 cycles
The type of DNA polymerase used is very important

Taq DNA polymerase – isolated from a species known

as Thermus aquaticus that thrives in hot springs

Why can't you use DNA Polymerase isolated from

bacteria that live at 37 C?

Answer: the enzyme would be denatured

during all 3 PCR temperatures
 Polymerase Chain Reaction (PCR)

Ch3 Figure 6 (textbook)

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 Advantage of PCR

• Can amplify millions of copies of target DNA from small

amount of starting material in short period of time
• To calculate the number of copies of target DNA
starting with 1 molecule of DNA use this equation 2N in
which N represents number of PCR cycles
• Assume you want to do 22 PCR cycles to amplify your
DNA insert, how many copies of DNA will you have at
the end of your PCR?

Answer: 222 = 4,194,304

Can amplify millions of copies of target DNA from small
amount of starting material in short period of time
To view results of PCR use a technique known as
gel electrophoresis

DNA can be seen

under UV light
 Applications of PCR
• Making DNA probes
• Studying gene expression
• Detection of viral and bacterial infections
• Diagnosis of genetic conditions
• Detection of trace amounts of DNA from tissue found at crime
scene
• Detection of DNA from fossilized dinosaur tissue
 Polymerase Chain Reaction (PCR)

Ch3 Figure 7 (textbook)

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 3.4 Laboratory Techniques and Applications
of Recombinant DNA Technology

Agarose Gel Electrophoresis

Separate and visualize DNA fragments based on size
 What is agarose?
• Agarose is isolated from seaweed

• when melted in a buffer solution and poured into a

horizontal tray and as it cools it will form a semisolid gel
containing small pores through which DNA will travel
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
 Agarose Gel Electrophoresis

• To run a gel, it is submerged in a buffer solution that

conducts electricity

• DNA is loaded into small depressions called wells at the top

of the gel

• Electric current is applied through electrodes at opposite

ends of the gel
DNA migrates according to its charge and size
• DNA molecule is negatively charged

--- Rate of migration through the gel depends on the

size of the DNA because the sugar phosphate
backbone makes it always negatively charged

----DNA migrates toward positive pole and is repelled

by negative pole

• Migration distance is inversely proportional to size of

DNA fragment

• Large fragments migrate slowly

• smaller fragments migrate faster
DNA migrates according to its charge and size
 Agarose Gel Electrophoresis

---Tracking dye is added to the samples to monitor

DNA migration during electrophoresis
 Agarose Gel Electrophoresis

---DNA can be visualized after electrophoresis by the

addition of DNA staining dyes
Ethidium bromide: intercalate between DNA base pairs and
it fluoresces under ultraviolet light
Then a picture can be taken to document the
gel results
 Example of gel electrophoresis result

Marker DNA Samples A,B,C

basepairs
2000 A
1500
B
1000
C
500

• Here is a result in which you ran your DNA fragments on a 1% gel. What is
the approximate size of band B? Why did it not migrate super fast through
the gel?
Agarose Gel Electrophoresis

Ch3 Figure 10 (textbook)

Chapter Contents
• 3.1 Introduction to Recombinant DNA
Technology and DNA Cloning
• 3.2 What Makes a Good Vector?
• 3.3 How Do You Identify and Clone a Gene of Interest?
• 3.4 Laboratory Techniques and Applications of
Recombinant DNA Technology
• 3.5 Genomics and Bioinformatics: Hot
Disciplines of Biotechnology
 Southern blot analysis
a method used in molecular biology for
detection of a specific DNA sequence in DNA
samples

[Link]
 Uses of southern blot

• Used to determine gene copy number

• gene mapping
• gene mutation detection
• PCR product confirmation
• DNA fingerprinting
 Procedures

• Digest chromosomal DNA into small fragments with restriction enzymes

• Fragments are separated by agarose gel electrophoresis
• Gel is treated with alkaline solution to denature the DNA
• Fragments are transferred onto a nylon or nitrocellulose filter (called
blotting)
• Filter (blot) is baked or exposed to UV light to permanently attach the DNA
• Filter (blot) is incubated with a labeled probe and exposed to film by
autoradiography
• Number of bands on film represents gene copy number

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Procedures of southern blot
southern blot
 Example of Southern blot result

18

Samples NT and 18: DO NOT have the gene

Samples P, 19, 25: have one copy of the gene
Samples 6, 12, 33: have two copies of the gene
Sample 17: have three copies of the gene
Southern blot

Ch 3 Figure 15 (textbook)
 Studying Gene Expression

Techniques involve analyzing mRNA produced by a tissue

1- Northern blot analysis

2- Reverse transcription PCR

3- Real time or quantitative (qPCR)

4- Gene microarrays
 1- Northern blot analysis

Technique involve
analyzing mRNA
produced by a
tissue
 1- Northern blot analysis

• Basic method is similar to Southern blotting

• RNA is isolated from a tissue of interest, separated by

gel electrophoresis, blotted onto a membrane, and
hybridized to a labeled RNA probe, exposed bands on
autoradiograph show presence of mRNA for gene of
interest as well as size of mRNA

• Can compare and quantify amounts of mRNA present

in different tissues
Northern blot procedure
 2- Reverse transcription PCR

Used to study mRNA levels when level of detection is

below that of Northern blot

Start with mRNA to make cDNA

 What is the the different between PCR from last
lecture and reverse transcriptase PCR?
The starting sample is the only difference, everything else
the same

Starting sample is different

Regular PCR Reverse transcriptase PCR
Genomic DNA sample mRNA sample (ONLY exons)
(introns+exons) then we make cDNA

Everything else is the same

Everything else is the same
 4- Gene microarrays
enable researchers to study all of the genes
expressed in a tissue very fast

Gene microarrays or DNA microarrays (gene chip)

is created with use of small glass microscope slide
 Very common usage of DNA microarray

• Common to do microarray in which you compare two

different conditions with 2 different colored dyes (one for
treatment and other for control condition)

• Laser is scanned at different wavelengths for each probe

and then the images are overlaid to make direct
comparisons between the treatment and control

• Example study gene expression difference between cancer cells

and normal cells to look for genes possibly involved in cancer
progression
• Results of such studies can possibly lead to new drug therapies to
combat cancer and other diseases
 DNA Sequencing
5. DNA sequencing
 DNA Sequencing
The sequence of nucleotides of any gene/genes
Three main methods of DNA sequencing techniques

1- Chain termination sequencing (Sanger method)

2- Shotgun method

3- Next-Generation Sequencing (NGS)