18/12/20

Recombinant DNA
Technology

Needles in Haystacks
• How to find one gene in large genome? A gene
might be 1/1,000,000 of the genome. Three
basic approaches:
• 1. Polymerase chain reaction (PCR). Make
many copies of a specific region of the DNA.
• 2. cell-based molecular cloning: create and
isolate a bacterial strain that replicates a copy of
your gene.
• 3. hybridization: make DNA single stranded,
allow double strands to re-form using a labeled
(e.g. radioactive) version of your gene to make it
easy to detect.

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Polymerase Chain Reaction

• Based on DNA polymerase creating a second strand of DNA.
– Needs template DNA and two primers that flank the region to be
amplified. Primers are short (generally 18-30 bases) DNA
oligonucleotides complementary to the ends of the region being
amplified.
– DNA polymerase adds new bases to the 3' ends of the primers to create
the new second strand.
– go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA from this
region
– A key element in PCR is a special form of DNA polymerase from
Thermus aquaticus, a bacterium that lives in nearly boiling water in the
Yellowstone National Park hot springs. This enzyme, Taq polymerase,
can withstand the temperature cycle of PCR, which would kill DNA
polymerase from E. coli.
• advantages:
– rapid, sensitive, lots of useful variations, robust (works even with partly
degraded DNA)
• disadvantages:
– Only short regions (up to 2 kbp) can be amplified.
– limited amount of product made

PCR Cycle
• PCR is based on a cycle of 3
steps that occur at different
temperatures. Each cycle
doubles the number of DNA
molecules: 25-35 cycles
produces enough DNA to see
on an electrophoresis gel.
Each step takes about 1 minute
to complete.
• 1. Denaturation: make the
DNA single stranded by
heating to 94oC
• 2. Annealing: hybridize
the primers to the single
strands. Temperature varies
with primer, around 50oC
• 3. Extension: build the
second strands with DNA
polymerase and dNTPs: 72oC.

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Other PCR Images

DNA Amplification in PCR

• original DNA: very long
molecules with neither end well
defined. Number stays
constant in the PCR reaction:
no new ones are made.
• initial PCR product made from
original DNA: has one end
defined by the primer, but the
other end is not well defined.
Copy number grows linearly.
• all other PCR products have 2
ends defined by the primers,
so they have a constant length
and can be easily detected by
electrophoresis. Copy number
grows exponentially.

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Electrophoresis
• Separation of charged molecules
in an electric field.
• Nucleic acids have 1 charged
phosphate (- charge) per
nucleotide. means constant chare
to mass ratio. Separation based
(mostly) on length: longer
molecules move slower.
• Done in a gel matrix to stabilize:
agarose or acrylamide.
• average run: 100 Volts across a
10 cm gel, run for 2 hours.
• Stain with ethidium bromide:
intercalates between DNA bases
and fluoresces orange.
• Run alongside standards of known
sizes to get lengths

PCR Applications
• RT-PCR: use reverse
transcriptase to convert
messenger RNA into DNA,
then amplify it with PCR.
• Anchor-primed PCR: use one
sequence-specific primer and
use a set of random primers
for the other end. For
example: 3’ RACE-PCR
(Rapid Amplification of cDNA
Ends) uses an oligo-dT primer
to bind to the poly A tail of
mRNA and a universal primer
for the internal region.
• Adding linkers to the primers
puts them into the amplified
DNA. Useful for cloning or
further PCR.

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Allele-Specific PCR
• For base change
mutations (single
nucleotide
polymorphisms).
• Use a primer
whose 3’ base
matches the
mutation. Will
amplify one allele
but not the other
because the 3’ end
is not paired with
the template in the
wrong allele.

SSR Genetic Markers

• . Microsatellites (Simple Sequence Repeats: SSRs). Used for mapping the human
genome--the main marker system used today.
• SSRs are short (2-5 bases) sequences that are repeated several times in tandem:
TGTGTGTGTGTG is 6 tandem repeats of TG.
• SSRs are found in and near many genes throughout the genome--they are quite
common and easy to find.
• During normal replication of the DNA in the nucleus, DNA polymerase sometimes
slips and creates extra copies or deletes a few copies of the repeat.
• This happens rarely enough that most people inherit the same number of repeats that
their parents had (i.e. SSRs are stable genetic markers), but often enough that
numerous variant alleles exist in the population.
• Mapping SSRs is a matter of having PCR primers that flank the repeat region, then
examining the PCR products on an electrophoresis gel and counting the number of
repeats.
• SSRs are co-dominant markers: both alleles can be detected in a heterozygote.

• If an SSR is a 3 base repeat within the coding region of a gene, it will create a
tandem array of some amino acid. Certain genetic diseases, most notably
Huntington's Disease, are caused by an increase in the number of repeats: once the
number gets high enough the protein functions abnormally, causing neural
degeneration. Such SSRs are called "tri-nucleotide repeats" or TNRs.

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SSR Example

Cell-Based Molecular Cloning

• The original recombinant DNA technique: 1974 by Cohen and
Boyer.
• Several key players:
• 1. restriction enzymes. Cut DNA at specific sequences. e.g.
EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC.
– Used by bacteria to destroy invading DNA: their own DNA has been
modified (methylated) at the corresponding sequences by a methylase.
• 2. Plasmids: independently replicating DNA circles (only circles
replicate in bacteria). Foreign DNA can be inserted into a plasmid
and replicated.
– Plasmids for cloning carry drug resistance genes that are used for
selection.
– Spread antibiotic resistance genes between bacterial species
• 3. DNA ligase. Attaches 2 pieces of DNA together.
• 4. transformation: DNA manipulated in vitro can be put back
into the living cells by a simple process .
– The transformed DNA replicates and expresses its genes.

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Plasmid Vectors
• To replicate, a plasmid must be
circular, and it must contain a replicon,
a DNA sequence that DNA
polymerase will bind to and initiate
replication. Also called “ori” (origin of
replication).
– Replicons are usually species-specific.
– Some replicons allow many copies of
the plasmid in a cell, while others limit
the copy number or one or two.
• Plasmid cloning vectors must also
carry a selectable marker: drug
resistance. Transformation is
inefficient, so bacteria that aren’t
transformed must be killed.
• Most cloning vectors have a multiple
cloning site, a short region of DNA
containing many restriction sites close
together (also called a polylinker).
This allows many different restriction
enzymes to be used.
• Most cloning vectors use a system for
detecting the presence of a
recombinant insert, usually the blue/
white beta-galactosidase system.

Basic Cloning Process

•Plasmid is cut open with a restriction
enzyme that leaves an overhang: a
sticky end
•Foreign DNA is cut with the same
enzyme.
•The two DNAs are mixed. The sticky
ends anneal together, and DNA ligase
joins them into one recombinant
molecule.
•The recombinant plasmids are
transformed into E. coli using heat
plus calcium chloride.
•Cells carrying the plasmid are
selected by adding an antibiotic: the
plasmid carries a gene for antibiotic
resistance.

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DNA Ligase in Action!

I hope

Cloning Vector Types

• For different sizes of DNA:
– plasmids: up to 5 kb
– phage lambda (λ) vectors: up to 50 kb
– BAC (bacterial artificial chromosome): 300 kb
– YAC (yeast artificial chromosome): 2000 kb
• Expression vectors: make RNA and
protein from the inserted DNA
– shuttle vectors: can grow in two different
species

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Lambda-based vectors
• Phage lambda can do 2 different things
when it enters the cell:
– lytic cycle: it can start reproducing itself
immediately, which produces about 200 new
phage in 15 minutes and kills the cell
– lysogenic cycle: the lambda DNA can
integrate into the host chromosome and
remain dormant for many generations. When
given the proper signal, the integrated DNA
(prophage) leaves the chromosome and
enters the lytic cycle.
• Lambda is about 50 kb long, and the central
20 kb is only used for lysogeny; it can be
replaced by foreign DNA.
– Ligation of arms with insert using DNA ligase
– Packaged into phage particles in vitro using
extracts from cells that have contain pieces of
the phage heads.
– Use these phage to infect new E. coli.

• Cosmids are similar to phage vectors: use

lambda, but remove all but the ends (cos
sites), ori, and selectable marker. Package
in vitro--becomes a large (50 kb) plasmid in
the E. coli.

Bacterial Artificial Chromosomes

• Based on the F plasmid that
confers the ability to conjugate.
• Low copy number plasmids
(usually 1 per cell), which prevents
crossing over between repeated
sequences in the insert DNA
• But, low copy number also means
low DNA yield.
• Transformed into E. coli using
electroporation, subjecting the
bacteria to a high voltage
electrical field.

• BACs are currently the most

common vector for large inserts
such as eukaryotic genome
projects.

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Yeast Artificial Chromosomes

• A linear chromosome, has
centromere, telomeres, ARS
(autonomously replicating
sequence), selectable marker
for yeast (uracil or tryptophan
biosynthesis genes usually).
• Also has E. coli ori and
selectable marker: you can
grow the vector itself in E. coli
• Then purify it, ligate in foreign
DNA, transform into yeast.

Expression Vectors
• Various types:
– RNA only: use a vector that has a phage T7 promoter in front of
the cloning site, and an inducible T& polymerase gene.
Induction by the lac operon repressor gene and the synthetic
inducer IPTG (isopropyl thiogalactoside).
– polypeptide or fragments of polypeptides: can be produced in E.
coli using a ribosome binding site in addition to the promoter.
Need to use the correct reading frame.
• can also be done as a fusion protein (your protein fused to a marker
protein) for easy detection or purification
– post-translationally modified or intron-spliced protein: needs to
be expressed in eukaryotic cells. Needs eukaryotic promoter and
polyadenylation (poly-A addition) signals, plus a selectable
marker that works in eukaryotes (since most antibiotics are
specific for prokaryotes).

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Example Expression Vector

• For eukaryotic expression, this vector (from
Invitrogen) has a cauliflower mosaic virus
promoter (PCMV), a bovine growth hormone
polyadenlyation site (BGHpA).
• The DNA inserted at “hORF” gets fused to
a short peptide called an epitope, for which
very specific anitbodies exist. It also gets
fused to 6 histidines, which allow easy
purification on a column that has nickel ions
bound to it (an affinity tag).
• For growth in mammalian cells, it has an
SV40 viral origin of replication (SV40ori),
and a zeocin resistance gene (Zeocin, with
SV40 promoter/enhancer and SV40 poly A
site).
• For growth in E. coli it has the ColE1
replicon. Zeocin works as a selectable
marker in baceria as well as in eukaryotic
cells.
• There is also a T7 promoter for making RNA
from the inserted gene, and an f1 origin of
replication for making single stranded DNA
(useful for sequencing).

Sources of DNA to Clone

• Genomic DNA: cut up whole genome and clone small pieces.
Advantage is, you get everything. Disadvantage is, a lot of it is junk.
– Two general methods:
• 1. randomly shear DNA into small pieces, then ligate linkers to the ends:
oligonucleotides that contain a useful restriction site.
• 2. partially digest the DNA with a restriction enzyme that has a 4 base
recognition site. These sites will appear at random every 256 (44) base
pairs. Take long pieces.

• cDNA: DNA copy of mRNA, made with reverse transcriptase.

Advantage: you just get the expressed genes. Disadvantages: you
don't get control sequences or introns, and frequency depends on
level of expression.

• Synthetic DNA: synthesized de novo (for example multiple cloning

sites or linkers), or made by PCR

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