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Chapter 8:

Recombinant DNA
Technology and
Molecular Cloning

Sometimes a good idea comes to you when you are not


looking for it. Through an improbable combination of
coincidences, naivet and lucky mistakes, such a
revelation came to me one Friday night in April, 1983, as
I gripped the steering wheel of my car and snaked along
a moonlit mountain road into northern Californias
redwood country. That was how I stumbled across a
process that could make unlimited numbers of copies of
genes, a process now known as the polymerase chain
reaction (PCR)
Kary B. Mullis, Scientific American (1990), 262:36

8.1 Introduction

The cornerstone of most molecular biology


technologies is the gene.
To facilitate the study of a genes:
Clone the gene by inserting it into another
DNA molecule that serves as a vehicle or
vector that can be replicated in living cells.

When two DNAs (the insert and vector) of


different origin are combined, the result is a
recombinant DNA molecule.
The recombinant DNA is placed in a host cell,
amplified, and purified for further analysis.

8.2 The beginnings of


recombinant DNA technology

Recombinant DNA technology arose


through the efforts of several research
groups working primarily on
bacteriophage lambda ().

Insights from bacteriophage lambda ()


cohesive sites
In 1962, Allan Campbell noted that the linear
genome of bacteriophage forms a circle upon
entering the host bacterial cell by joining
complementary single-stranded DNA cohesive
(cos) sites.
The idea of joining DNA segments by cohesive
sites became a guiding principle in the
development of recombinant DNA technology.

Insights from bacterial modification and


restriction systems
Salvador Luria and other phage workers
made the following observations:
Phages grown in one bacterial host fail to grow
in a different restrictive bacterial host.
The phage DNA is degraded in the restrictive
host.

Rare progeny phages become modified in


some way so that they grow normally in the
new host.
The modification was reversible.
1962: The molecular basis of restriction and
modification was defined by Werner Arber and
coworkers.

Restriction system
Restriction endonucleases
First restriction endonuclease characterized in
E. coli K-12 by Matt Meselson and Bob Yuan.
Restrict or prevent viral infection by
degrading the invading nucleic acid.

Modification system
Methylase activity: Addition of methyl groups to
protect those sites in DNA sensitive to attack
by a restriction endonuclease.
Typically adenine methylation (6-methyl
adenine).
Methylation pattern is maintained during DNA
replication.

The first cloning experiments


One of the first recombinant DNA molecules
was a hybrid of phage and the SV40
mammalian DNA virus genome.
1974: first eukaryotic gene was cloned.
Amplified ribosomal RNA (rRNA) genes from
Xenopus laevis oocytes were cloned into a
bacterial plasmid.
The cloned frog genes were actively
transcribed into rRNA in E. coli.

I was tempted then to put together a book called


the Whole Risk Catalogue. It would contain risks
for old people and young people and so on. It
would be a very popular book in our semiparanoid society. Under D I would put dynamite,
dogs, doctors, dieldrin [an insecticide] and DNA. I
must confess to being more frightened of dogs.
But everyone has their own things to worry about.
James Watson, Genetics and Society (1993)

Fear of recombinant DNA molecules


1975: Recommendations from a landmark
meeting of molecular biologists formed the basis
for official guidelines developed by the National
Institutes of Health (NIH).
Activities involving the handling of recombinant
DNA and organisms must be conducted in
accordance with the NIH guidelines.
Four levels of risk are recognized, from minimal
to high.

8.3 Cutting and joining DNA

Two main categories of enzymes are


important tools in the preparation of
recombinant DNA
DNA ligases: join two pieces of DNA by forming
phosphodiester bonds.
Restriction endonucleases: recognize a
specific, rather short, nucleotide sequence on a
double-stranded DNA molecule, called a
restriction site, and cleave the DNA at this site
or elsewhere.

Major classes of
restriction endonucleases
Type II restriction endonucleases are
widely used by molecular biologists.
>240 available commercially.
6 bp cutters are the most commonly used.

Restriction endonucleases are named for


the organism in which they were
discovered:

HindIII from Haemophilus influenza (strain d)


SmaI from Serratia marcescens
EcoRI from Escherichia coli (strain R)
BamHI from Bacillus amyloliquefaciens (strain
H)

Recognition sequences for type II


restriction endonucleases
Orthodox type II restriction endonucleases
function as homodimers.
Recognition sequences are typically
palindromes.
Some enzymes generate sticky ends.
Some enzymes generate blunt ends.

Restriction endonucleases exhibit a great


degree of sequence specificity.
A single base pair change in the
recognition site eliminates enzymatic
activity.

The steps involved in restriction


endonuclease DNA binding and
cleavage
1.The first contact is nonspecific binding:
Interaction with the DNA sugar-phosphate
backbone only.
Catalytic center kept at a safe distance.

2. Random walk:
Sliding over short distances of <30-50
bp to target restriction site.
Hopping or jumping over longer
distances.
3. Specific binding at restriction site:

Large conformational change of the enzyme


and DNA (coupling).
Activation of catalytic center.

EcoRI: kinking and cutting DNA

Common structural core of four conserved strands and one -helix.

Large conformational change in EcoRI and the


DNA upon specific binding.

A central kink in the DNA brings the critical


phosphodiester bond between G and A deeper
into the active site and unwinds the DNA.

In the presence of Mg2+, EcoRI cleaves the


DNA on both strands at the same time to give
free 5-phosphate and 3-OH ends.

The exact mechanism by which cleavage


occurs has not yet been proven
experimentally.

DNA ligase joins linear pieces of DNA

The DNA ligase most widely used in the lab is


from bacteriophage T4.

T4 DNA ligase catalyzes formation of a


phosphodiester bond between the 5phosphate of a nucleotide on one fragment of
DNA and the 3-hydroxyl of another.

T4 DNA ligase will ligate fragments with sticky


ends or blunt ends, but for blunt ends the
reaction is less efficient.

To increase the efficiency of ligation,


researchers often use the enzyme terminal
deoxynucleotidyl transferase to modify the
blunt ends.

8.4 Molecular cloning

Basic molecular cloning procedure


1. DNA fragments to be cloned are generated
using restriction endonucleases.
2. Fragments are ligated to other DNA
molecules that serve as vectors.
3. Recombinant DNA molecules are transferred
to a host cell.
4. Cloned recombinant DNA is recovered from
the host cell for analysis.

Choice of vector is dependent on insert


size and application
Cloning vectors are carrier DNA molecules
with four important features:
1. Replicate independently.
2. Contain a number of restriction endonuclease
cleavage sites that are present only once.
3. Carry a selectable marker.
4. Relatively easy to recover from host cell.

The greatest variety of cloning vectors has


been developed for use in E. coli.
The first practical skill generally required
by a molecular biologist is the ability to
grow pure cultures of bacteria.

Classic cloning vectors:


Plasmids
Phages
Cosmids

New generation vectors:


Bacterial artificial chromosomes (BACs)
Yeast artificial chromosomes (YACs)
Mammalian artificial chromosomes (MACs)

Plasmid DNA as a vector


Plasmids are named with a system of uppercase
letters and numbers, where the lowercase p
stands for plasmid.
Low copy number plasmids: replicate to yield
only one or two copies in each bacterial cell.
High copy number plasmids: replicate to yield
>500 copies per bacterial cell.

Plasmid vectors are modified from naturally


occurring plasmids
Contain a specific antibiotic resistance gene.
Contain a multiple cloning site.

Five major steps for molecular cloning using


a plasmid vector
1. Construction of a recombinant DNA molecule.
2. Transfer of ligation reaction products to host
bacteria.
3. Multiplication of plasmid DNA molecules.
4. Division of host cells and selection of
recombinant clones, e.g. by blue-white
screening.
5. Amplification and purification of recombinant
plasmid DNA.

Transformation: transfer of recombinant


plasmid DNA to a bacterial host
Bacterial cells are incubated in a concentrated
calcium salt solution to make their membranes
leaky.
The permeable competent cells are mixed with
DNA to allow DNA entry.
Alternatively, a process called electroporation
drives DNA into cells by a strong electric current.

Why isnt the introduced foreign plasmid


DNA degraded by a bacterial restrictionmodification system?

Recombinant selection
Antibiotic resistance selects for transformed
bacterial cells.
Numerous cell divisions of a single transformed
bacteria result in a clone of cells visible as a
bacterial colony on an agar plate.
Successfully transformed bacteria will carry
either recombinant or nonrecombinant plasmid
DNA.

Blue-white screening

In the case of the vector pUC18, bluewhite screening is used to distinguish


recombinant from nonrecombinant
transformants.
Also known as lac selection or complementation

-galactosidase activity can be used as an


indicator of the presence of foreign DNA
If the lacZ 5 region of pUC18 is not interrupted by
inserted foreign DNA, the amino-terminal portion
of -galactosidase is synthesized.
The mutant E. coli host encodes only the carboxyl
end of -galactosidase.

The N-terminal and C-terminal fragments come


together to form a functional enzyme.
-galactosidase activity can be measured using
a colorless chromogenic substrate called X-gal.
Cleavage of X-gal produces a blue-colored
product, visualized as a blue colony on an agar
plate.
If a foreign insert has disrupted the lacZ 5 coding
sequence, X-gal is not cleaved and the bacterial
colonies remain white.

Amplification and purification of recombinant


plasmid DNA
Further screening to confirm the presence and
orientation of the insert.
Amplify positive (white) colony containing
recombinant plasmid DNA in liquid culture.
Purify plasmid DNA from crude cell lysates, e.g.
by chromatography and ethanol precipitation.

Liquid chromatography
Molecules dissolved in a solution will interact
(bind and dissociate) with a solid surface.
When the solution is allowed to flow across the
surface, molecules that interact weakly with the
solid surface will spend less time bound to the
surface and will move more rapidly.
Commonly used to separate mixtures of nucleic
acids and proteins.

Three main techniques


Gel filtration chromatography: separation by
differences in mass.
Ion-exchange chromatography: separation by
differences in charge.
Affinity chromatography: separation by
differences in binding affinity.

Bacteriophage lambda () as a vector


Phage vectors are particularly useful for
preparing genomic libraries.
The recombinant viral particle infects bacterial
host cells in a process called transduction.
Progeny viral particles appears as a clear spot of
lysed bacteria or plaque on a lawn of bacteria.

Artificial chromosome vectors


Bacterial artificial chromosomes (BACs) and
yeast artificial chromosomes (YACs) are
important tools for mapping and analysis of
complex eukaryotic genomes.
1997: first prototype mammalian artificial
chromosome (MAC)

Yeast artificial chromosome


(YAC) vectors
YAC vectors are designed to act like
chromosomes in host yeast cells
Origin of replication (Autonomously replicating
sequence, ARS)
Centromere
Telomere

YAC vectors contain selectable markers


URA3: encodes an enzyme required for uracil
biosynthesis.
TRP1: encodes an enzyme required for tryptophan
biosynthesis.
SUP4: tRNA that suppresses the Ade2-1 UAA mutation.

Red-white selection
Host yeast strain: ura3/trp1/Ade2-1 mutant
When foreign DNA is inserted in the multiple
cloning site, SUP4 expression is interrupted.
The Ade2-1 mutation is no longer suppressed.
ADE1 and ADE2 encode enzymes involved in
adenine biosynthesis.

Ade2-1 mutant cells produce a red pigment from


polymerization of an intermediate compound.
In the absence of foreign DNA, SUP4 is
expressed.
The Ade2-1 mutation is suppressed.
Ade2-1 mutant cells expressing SUP4 are white
(the color of wild-type yeast cells).

Sources of DNA for cloning


Genomic DNA
Chemically synthesized oligonucleotides
Previously isolated clones: subcloning
Complementary DNA (cDNA)
Polymerase chain reaction (PCR)

Complementary DNA (cDNA) synthesis


Most eukaryotic mRNAs have a poly(A) tail.
The poly(A) region can be used to selectively
isolate mRNA from total RNA by affinity
chromatography.
The purified mRNA can then be used as a
template for synthesis of cDNA by reverse
transcriptase.

Are 53 sequences that appear in the


literature the first strand or second
strand of the double-stranded cDNA?

Polymerase chain reaction (PCR)


Basic requirements for in vitro DNA synthesis:
DNA polymerase
DNA template
Free 3-OH to get the polymerase started
dNTPs

Three steps of the reaction performed in an


automated thermal cycler
Denaturation of the template DNA (e.g. 95C).
Annealing of primers (e.g. 55-65C).
Primer extension by a thermostable DNA
polymerase (e.g. 72C).

Taq DNA polymerase from Thermus aquaticus is


the most popular enzyme.
Pfu DNA polymerase from Pyrococcus furiosus
has higher fidelity.
Are the primers made of RNA or DNA?

Constructing DNA libraries


Genomic library: A cloned set of DNA fragments
that represent the entire genome of an
organism.
cDNA library: A cloned set of the coding region
of expressed genes only; derived from mRNA
isolated from a specific tissue, cell type, or
developmental state.

Genomic library
Break DNA into manageable sized pieces (e.g.
15-20 kb for phage vectors) by partial
restriction endonuclease digest.
Purify fragments of optimal size by gel
electrophoresis or centrifugation techniques.
Insert fragments into a suitable vector.
For the human genome, approximately 106 clones are
required to ensure that every sequence is
represented.

cDNA library
Does a cDNA library included intron
sequences or gene regulatory regions?

8.5 Library screening and


probes

Nowadays, a DNA sequence of interest is more


likely to be isolated by PCR than by a library
screen.
In PCR, the pair of primers limits the
amplification process to the particular DNA
sequence of interest.
In contrast, a DNA library can be perpetuated
indefinitely in host cells and retrieved whenever
the researcher wants to seek out a particular
fragment.

A key element required to identify a gene


during library screening is the probe:
A probe is a nucleic acid (usually DNA) that has
the same or a similar sequence to that of a
specific gene or DNA sequence of interest.
The denatured probe and target DNA can
hybridize when they are renatured together.

Library screening involves basic principles


of nucleic acid hybridization
Double-stranded nucleic acids can undergo
denaturation.
Complementary single strands spontaneously
anneal to a nucleic acid probe to form a hybrid
duplex.
The nucleic acid probe can detect a
complementary molecule in a complex mixture
with exquisite sensitivity and specificity.

Types of DNA and RNA probes


Oligonucleotide probes: chemically synthesized
DNA probes: cloned DNAs
RNA probes (riboprobes): made by in vitro
transcription from cloned DNA templates

Heterologous probes
A probe that is similar to, but not exactly
the same as, the nucleic acid sequence of
interest.

Homologous probes
A probe that is exactly complementary to
the nucleic acid sequence of interest.
Examples include:
Degenerate probes
Expressed sequence tag (EST) based probes
cDNA probes

Use of degenerate probes: historical


perspective
Before the advent of genome sequence
databases, the classic method for designing a
probe relied on having a partial amino acid
sequence of a purified protein.
Traditionally protein sequencing was performed
by Edman degradation.
Today protein sequencing is more often
performed using mass spectrometry technology.

Unique EST-based probes


ESTs are partial cDNA sequences of about 200-400 bp.
A computer program applies the genetic code to
translate an EST into a partial amino acid sequence.
If a match is found with the protein under study, the EST
provides the unique DNA sequence of that portion of
cDNA.
A probe can then be synthesized and used to screen a
library for the entire cDNA or genomic clone.

Using an identified cDNA to locate a


genomic clone
Use of a cDNA to locate a genomic clone
provides a highly specific probe for the
gene of interest.

Labeling of probes
A probe must be labeled, i.e. chemically
modified in some way which allows it, and
anything it hybridizes to, to be detected.

Radioactive and nonradioactive


labeling methods
Detection techniques
Autoradiography
Geiger counter
Liquid scintillation counter
Phosphorimager

Nonradioactive labeling
Colorimetric or chemiluminescent signals
Examples:
Digoxygenin-conjugated nucleotides are detected with
an anti-digoxygenin antibody conjugated to an enzyme
or fluorescent dye.
Biotin-conjugated nucleotides are detected using
enzyme-conjugated streptavidin.

Nucleic acid labeling


Method depends on application:
Internal (uniform) labeling or end labeling?
Radioactive or nonradioactive?

Labeling involves DNA or RNA synthesis


reactions or other enzyme-mediated
reactions.

Some methods for labeling nucleic acids:


Random primed labeling
In vitro transcription
Klenow fill-in

Library screening
Five major steps for screening a cDNA library
cloned into plasmid vectors:
1. Bacterial colonies are transferred to a
nitrocellulose or nylon membrane.
2. Bacterial cells are lysed and DNA is denatured.
3. Labeled probe is added to the membrane.
4. Washed membrane is exposed to X-ray film.
5. Positive colonies are identified.

Transfer of colonies to a
DNA-binding membrane
Bacterial colonies (members of the library)
grown on an agar plate are transferred to
nitrocellulose or nylon membrane to make a
replica.
After lysis and denaturation, the DNA is
covalently bound by its sugar-phosphate
backbone and the unpaired bases are exposed
for complementary base pairing.

Colony hybridization
The hybridization step is performed at a
nonstringent temperature that ensures the probe
will bind to any clone containing a similar
sequence.
Higher stringency washes are performed to
remove nonspecifically bound probe.
Heteroduplex stability is influenced by the number
of hydrogen bonds between the bases and base
stacking hydrophobic interactions.

The shorter the duplex, the lower the GC


content, and the more mismatches there are, the
lower the melting temperature (Tm).
Hybridization temperature is calculated as
follows:
Tm = 49.82 + 0.41 (%G + C) (600/l)
where l is the length of the hybrid in base
pairs.

Detection of positive colonies


The resulting autoradiogram has a dark spot on
the developed film where DNA-DNA hybrids
have formed.
If the gene is large, it may be fragmented over
multiple clones.
The original plate is used to pick bacterial cells
with recombinant plasmids that hybridized to the
probe.

Screening of expression libraries


Expression libraries are made with a
cloning vector that contains the required
regulatory elements for gene expression.
Useful for identifying a clone containing a
cDNA of interest when an antibody to the
encoded protein is available.

8.6 Restriction mapping and


RFLP analysis

Restriction mapping
Restriction mapping provides a compilation
of the:
Number of restriction endonuclease cutting sites
along a cloned DNA fragment.
Order of restriction endonuclease cutting sites.
Distance between restriction endonuclease
cutting sites.

Important roles for restriction mapping, for


example:
Characterizing DNA.
Mapping genes.
Diagnostic tests for genetic disease.
Checking the orientation of the insert in a
recombinant DNA clone.

DNA and RNA electrophoresis


When charged molecules are placed in an
electric field, they migrate toward the
positive or negative electrode according to
their charge.
Nucleic acids are separated by
electrophoresis within a matrix or gel.

Types of gel electrophoresis


Agarose gel electrophoresis
Pulsed field gel electrophoresis (PFGE)
Polyacrylamide gel electrophoresis (PAGE)

Restriction fragment length


polymorphism (RFLP)
The existence of alternative alleles associated
with restriction fragments that differ in size from
each other.
Variable regions do not necessarily occur in
genes.
Function of most RFLPs in the human genome
is unknown.
Exception: sickle cell anemia RLFP

Diagnosis of sickle cell anemia by restriction


fragment length polymorphism (RFLP)
and Southern blot
A point mutation in the -globin gene has
destroyed the recognition site of the restriction
endonuclease MstII.
Affected individuals: larger restriction fragment
on a Southern blot
Normal individuals: shorter restriction fragment

RFLPs can serve as markers of


genetic disease
A RFLP that is close to a disease gene tends to stay
with that gene during crossing-over (recombination)
during meiosis.
Linkage: the likelihood of having one marker
transmitted with another through meiosis.
When a PCR assay for typing a particular locus is
developed, it is generally preferable to RFLP analysis.

Southern blot

Method developed by Edward Southern.


Identify a specific gene fragment from the
often many bands on a gel.

PCR-RFLP assay for


maple syrup urine disease
Autosomal recessive disease: 1/176 in certain
Old Order Mennonite communities.
Missense mutation in one of the genes encoding
an enzyme involved in metabolism of branchedchain amino acids.
Tyrosine (Y) to asparagine (N) substitution:
Y393N allele.

Symptoms appear 4 to 7 days after birth.


Accumulation of -keto acid derivatives gives
urine a maple syrup-like odor.
Neurological deterioriation and death within 2 to
3 weeks if diet is not controlled.

PCR-RFLP assay to identify Y393N allele

Buccal swab or blood sample.


PCR
Cut PCR products with ScaI.
Agarose gel electrophoresis.
Stain with ethidium bromide.

8.7 DNA sequencing

DNA sequencing is the ultimate


characterization of a cloned gene.
Manual sequencing by the Sanger dideoxy
DNA method.
Automated DNA sequencing.
Next-generation sequencing.

Manual sequencing by the Sanger


dideoxy DNA method
Another DNA synthesis reaction
DNA polymerase (T7 DNA polymerase called
Sequenase)
DNA template
Free 3-OH to get the polymerase started
dNTPs

If the sequence is unknown how is the


primer designed?
Why is a ddNTP a replication terminator?

Automated DNA sequencing


Developed by Leroy Hood and Lloyd Smith in
1986.
Each ddNTP terminator is tagged with a different
color of fluorophore.
DNA samples loaded in a capillary array migrate
through a gel matrix by size, from smallest to
largest.

Automated DNA sequencing


When DNA fragments reach the detection
window, a laser beam excites the fluorophores
causing them to fluoresce.
An electropherograma graph of fluorescence
intensity versus timeis converted to the DNA
sequence by computer software.

Next-generation sequencing
The sequencing of spatially separated, clonally
amplified DNA templates in a massive array all
at the same time.
DNA sequences in the range of hundreds of
megabases to gigabases can be rapidly
obtained.

454 pyrosequencing
Individual nucleotides are detected by light
production as nascent DNA is synthesized
one nucleotide at a time.

Template DNA is prepared by emulsion PCR.


The template DNA is immobilized on a bead in a
well in the sequencing machine.
Solutions of A,C,G, and T nucleotides are
sequentially added and removed from the
reaction.
The enzyme luciferase is used to generate light.
Light is only produced when the nucleotide
solution complements the first unpaired base of
the template.