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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, naiveté 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 California’s
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 5′phosphate 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 isn’t 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 5′→3′ 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. 95C).
• Annealing of primers (e.g. 55-65C).
• Primer extension by a thermostable DNA
polymerase (e.g. 72C).
• 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 electropherogram―a graph of fluorescence
intensity versus time―is 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.