Recombinant DNA

1
Recombinant DNA Techniques

• Three Parts
• DNA purification, isolation, and sequencing
• Tools for analyzing gene structure and expression
• Genome mapping linking genetic and physical maps

2
DNA Purification

• Extraction of DNA Simple
• Lyse Cells (e.g., chemicals, enzymes, etc)
• Spin Down Cell Debris
• Remove Proteins
• phenol extraction
• Ion-exchange columns
• CsCl Gradients
• glass beads with affinity for DNA
• Concentrate DNA Ethanol Precipitation
• Treat with ribonucleases to remove RNA
• DNA is now pure but too complex to study.
• Thousands to millions to billions of base pairs.

DNA H20
proteins phenol
3 vol EtOH
DNA H20
mix, spin, collect DNA pellet
3
Historically, early studies were done on
organisms with relatively small amounts of DNA
(genomes) OR small elements of DNA separable the
the rest of the genomic DNA

• rDNA from Xenopus or Tetrahymena.
• - Genes for rRNA
• Mitochondrial DNA (mtDNA)
• Chloroplast DNA
• Plasmid
• Viruses and Phages

• many copies in a cell
• all copies the same size
• can be purified easily (e.g., by sucrose
  gradient)

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Tools For Study ofDNA Structure Organization
I. Electrophoresis Separating DNA based on size
by driving it through a gel matrix via the
application of a current (-) anode ? DNA (-
charge) ? () cathode Shorter DNA (or RNA)
fragments move through the gel faster than long
fragments - some exceptions, folded structure can
alter mobility of DNA though a gel. (e.g.,
supercoiled vs. relaxed plasmids)
Two Types of Gels 1. Acrylamide single base
resolution i,e., for sequencing gels 2. Agarose
separation of large fragments i.e., for plasmids
5
Agarose Gel Stained with ethidium bromide (EtBR)
to Visualize the DNA
slots where DNA is loaded
1000 bp
700 bp
600 bp
500 bp
Screening PCR products to test for the presence
of specific DNA sequences
molecular weight markers
molecular weight markers
correct PCR product
6

• Restriction Enzymes
• endonucleases that recognize and cut specific DNA
  sequences
• Target sequences of these enzymes are palindromic
  (result of these enzymes being homodimers)
• Palindromic sequences are where both strands are
  the same sequence. i.e.,
• Commonly recognize 4 or 6 base-pair targets
• Cuts on average, every 256 or 4096 bp

5-ATAT-3 3-TATA-5
Mbo I (4 cutter)
5--AACGATCGAA-- --TTGCTAGCTT--
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• while many restriction enzymes leave sticky
  ends, some are blunt end cutters.
• enzymes that look for 8, 10 , 12 base-pair
  targets are also known Some of these allow a
  certain amount of degeneracy / asymmetry in their
  target sequence

EcoRV 5-GATATC- -CTATAG-5
-AGACGTCT- C A
The enzyme is less specific because it will cut
at any of these so-called degenerate sequences
8

• DNA Ligase(s)
• forms phosphodiester bonds
• reverse action of endonucleases
• Require ATP and a 5 phosphate on the 3
  substrate strands
• work more efficiently when sticky ends overlap
  but can also repair, or join, blunt ends

OH (P)
OH
P
ATP Ligase
5-AATCGATCGTCC- -TTAGCTAGCAGG-5
5-AATCGATCGTCC- -TTAGCTAGCAGG-5
P
OH
- the enzyme is not sequence-specific except for
the fact that sticky ends will ligate more easily
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• Kinases and Phosphatases
• add or remove phosphate groups and the 5 ends of
  DNA or RNA.

Kinase ATP
PO4-GATC
HO-GATC
Phosphatase
gamma
alpha
beta
- the enzyme is not sequence-specific
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• Other Useful Enzymes
• Single Stranded and Double Stranded specific
  endonucleases
• RNA and DNA polymerases
• T7 RNA Polymerase DNA ? RNA
• Reverse Transcriptase RNA ? DNA
• Klenow Fragment
• Taq polymerase PCR

S1 nuclease Mung Bean Nuclease are ssDNA
specific nucleases
also RNaseH (cleaves RNA in RNA-DNA
hybrid), DNAse (destroys dsDNA), RNAse (destroys
RNA)
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• Plasmids
• along with restriction endonucleases, ligases,
  etc., they are the single most useful tool in
  molecular biology
• they often have a high copy number in a cell, and
  they can easily be purified away from chromosomal
  DNA
• one of the most common Vectors used in
  recombinant DNA techniques
• ? the molecule that is modified / altered to
  incorporate DNA sequences of interest and then
  re-introduced back into a host organism (usually
  bacteria)

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pBluescript
origin of replication
A widely used plasmid cloning vector
ampicillin resistance gene
MCS
MCS, Multiple Cloning Site
13
You are assigned sections 25A-25E (pp. 969-979)
in the Mathews text book, which cover various
techniques for recombinant DNA technologies
Creation of a Recombinant DNA molecule in
vitro See figure 25B of text
5 ...GAATTC...
...GAATTC... 3
3 ...CTTAAG...
...CTTAAG... 5
Eco RI
Eco RI
Separation of restriction fragments
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Figure 25.6
Agarose gel stained with EtBr, allows you to
visualize all of the fragments. If 32P
end-labels were used to label the intact molecule
to visualize the bands by autoradiography, only
the terminal fragments would be observed.
15
An example of cloning of random genomic
fragments start with a plasmid that carries
genes for resistance for two antibiotics.
A
Cut with restriction enzyme A
Plasmid
A
Mix Ligate
wild type
Genomic DNA
Resistant to both antibiotics.
Recombinant DNA
Transform
Resistant to ONE Susceptible to the other
antibiotics
Susceptible to both Antibiotics
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Selecting for Transformed Cells by Replica-plating
- cannot select the recombinant DNA directly
because the efficiency of successful insertion is
very low and the product cannot be visualized or
detected -All the sequences are transformed and
we select for the cells which contain the
plasmids of interest - looking for those
resistant to one antibiotic and sensitive to the
other
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• The DNA into which a foreign piece of DNA is
  cloned is called a VECTOR
• There are several classes of vectors in use
• Plasmids Accept up to 10 kb foreign DNA
• Phage ? 5-20 kb fragments (its own genome is
  only 50 kb!) Commonly used in making genomic
  libraries. (very high efficiency of transfection)
• Cosmids 35-45 kb similar to plasmids (high
  efficiency for transformations)
• YACs (Yeast Artificial Chromosomes) 300-2000 kb!
  (essential for cloning very large fragments)

18

• Types of Inserts
• Genomic DNA ? for making genomic libraries
• cDNA only expressed sequences, i.e., those that
  are transcribed into RNA no introns, only exons
• - the c in cDNA signifies complementary to RNA

19
Making cDNA
the reverse transcriptase is not highly
processive, so you end up with some
incompletely-synthesized first-strand DNA
normally RNAseH is used in this step
the 3 end of any particular ssDNA may not form
such a hairpin
one normally uses a mixture of random primers in
this step
this step is not necessary if you use random
primers
end result cDNA where not all molecules are
full-length
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Labeling Techniques

• Radiolabeling (easy, allows for very sensitive
  detection of nucleic acids)
• End Labeling - kinasing

5 HO
5 32P
32Pgamma-ATP Polynucleotide kinase

• Primer Extension Nick Translation
• Methods to introduce multiple labels in a molecule

32Palpha-dNTPs Klenow fragment, or DNA polymerase
often, random primers are used
(One strand is labeled unless you use
random primers, in which case both strands are
labeled)
21

• RNA Labeling sometimes it is desirable to use
  RNA polymerase and 32P-labeled NTPs to make
  labeled RNA molecules

• instead of radiolabeling, a variety of chemical
  / fluorescent labels can also be used to label
  DNA/RNA
• dNTPs conjugated with
• these groups are available

- biotin ? binds avidin VERY tightly -
fluorescein dye (fluorescent)
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Restriction Mapping - purify DNA - digest (cut)
with different restriction enzymes - run on an
agarose gel determine fragment size
2.5
A
B
1.5
1
10 kb (10000 bp)
B
A
2
3
A
- first step taken to create a road-map (genetic
map) with an unknown DNA (especially of large
sequences)
kb
6-
4.5----
Q what would you see if an enzyme cut only once
(at a so-called unique restriction site? Q what
would you see if you had not cut the plasmid?
4-
3.5----
2-
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Physical mapping
BamHI
1.2 0.4
A
electrophoresis
B
A. determining position of BamHI site B.
Determining the orientation of the HindIII
fragment in the vector using BamHI and EcoRI
enzymes
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Constructing a Genomic Library - extract genomic
DNA - cut with a restriction enzyme (want only
partial cutting) - mix with an excess of plasmid
cut with the same enzyme - ligate - transfer
(transform) into bacteria.
Pick a 4-cutter enzyme ie. Hae III ? AGCT Partial
Digestion
why would you only want partial cutting of the
DNA?
25
Select out pieces of 20 kbases long, by
electrophoresis
Digested DNA (smear, consisting of fragments of
many different sizes)
MW ladder
40 kb ?
clone into vector
30 kb ?
Make recombinant DNA in appropriate vector
-cut out use DNA from this region
20 kb ?
10 kb ?
5 kb ?
- you can obtain a collection of clones of
different sequences that include the entire
genome of the organism
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cDNA libraries can also be made from different
cell types, tissues, different developmental
stages, cells exposed to different conditions,
etc., in order to make libraries that contain
expressed sequences of ones choice
Clone these to make a library (or mixture) of
cDNAs Should contained all expressed
(transcribed) sequences
cDNA
genes
mRNA
Clone restriction fragments these to make a
library (or mixture) genomic DNA. Should contain
all genomic DNA sequences
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Polymerase Chain Reaction(PCR)

• A highly versatile technique that can be used for
  many purposes, Particularly, for DNA
  identification amplification/purification
• PCR is
• Extremely Fast
• Extremely Sensitive
• Requires only partial knowledge of DNA sequence
• Leads to exponential amplification of a selected
  DNA fragment
• Good for the isolation of known fragments from
  many individuals
• 1 Forensic analysis.
• Studies of relatedness
• Studies of molecular evolution
• Good for the detection of rare sequences
• Diagnostic tool for the detection of viruses and
  bacteria
• Good for the isolation of multi-gene families
• Use primers that recognize conserved regions

used in nearly all life science fields for
cloning, making different recombinant DNA
molecules
28
Denature at 94ºC
29
Site Directed Mutagenesis
see Figure 25E.1
- clone fragment to be modified into M13 (single
stranded DNA phage vector)
15-20 bases with a single mismatch or a mismatch
containing a few bp changes CAN MUTATE ONE OR
MORE CODONS
Collect phage and purify
M13
Want to make a mutation at this spot
DNA Pol, dNTPs Ligase
Transfect into bacteria
Mutant Gene
This can also be done using a PCR-based approach
Wild type
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PCR-based site directed mutagenesis
Inserty 5.22 here
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DNA Sequencing

• Two Methods
• Maxam-Gilbert Chemical Sequencing
• 1st method developed, but not used much now for
  sequencing ? still used for footprinting
• Sanger Enzymatic dideoxy Sequencing
• - Basic method used in all modern sequencing
  protocols
• - Uses a DNA polymerase, primer and dNTPs plus a
  small proportion of ddNTPs (chain terminators)

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• - The Sanger sequencing method is now automated
  and can be done by machine (robots). This is the
  method now used by virtually all sequencing
  projects.
• for example, Celera, a company that sequenced
  the human genome, has a facility with 100s of
  sequencers that work night and day -- generating
  3 billion bp of seq.
• - DNA sequencing allows inference of protein
  sequence (genetic code)
• - DNA or protein sequences can be compared to all
  other known sequences by computer using search
  algorithms
• - can give information about related proteins
• - may provide clues about structure function
  of newly-discovered protein

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Dideoxy-Nucleotides
Base
(P)-(P)-(P)-
O
H
OH
dNTP
ddNTP
If a ddNTP is added to a growing DNA chain, the
sequence is terminated, because another base can
not be added
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SANGER DIDEOXY SEQUENCING
primer
End labeled
ddNTP Reaction Mix
5-
DNA Polymerase dNTPs one of
4 tubes
C T A G
5 C G T A A G G T A T C C 3
ddATP
ddTTP
ddCTP
ddGTP
5-
C
5-
C
5-
autoradiograph of dried sequencing gel
C
In the ddGTP reaction there is a random chance a
ddGTP will be incorporated across from a C
Sequence is complementary to the strand being
sequenced!
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The dideoxy method has been modified so it can be
done in one tube. In this case all of the
ddNTPs are labeled with different, coloured
fluorescent molecules
This makes automation much simpler, reduces the
cost of the reactions, and speeds up the process
tremendously. Large sequencing centres use this
method and can read millions of bp every single
day
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Output of a DNA sequence from an automated
sequencer
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MAXAM-GILBERT CHEMICAL SEQUENCING
Labeled strand (P32)
ddNTP Reaction Mix
5-
Treat with chemicals that specifically cleave at
a certain base.
4 tubes
C T A G
3 G C A T T C C A T A G G 5
AgtG -rxn
TgtC -rxn
G-rxn
C-rxn
5-
C
5-
C
5-
C
autoradiograph of dried sequencing gel
The reaction is done such that each strand is
cleaved only once
Sequence is read directly
38
Dnase Footprinting
A method to detect where a protein binds to DNA.
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Blotting Techniques
Biomolecules can be fixed to membranes of filter
paper, then they can be assayed for binding to
various probes - Southern Blot Use a DNA
sequence to detect DNA - Northern Blot Use a DNA
sequence to detect RNA - Western Blot Use an
antibody to detect proteins - South-Western DNA
binding to proteins or vice versa Nucleic Acid
Nucleic Acid interactions are simply due to
Watson Crick base-pairing When done on a
membrane we call it hybridization.
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- temperature at which hybridization is done
is the most critical factor - hybridization
temperature should be high when the amount of
homology, or sequence similarity between probe
and template is high - conversely, hybridization
temperature should be low when homology is low
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Plaque hybridization
42
Antibody screening of ?GT11expression clones
43
Southern blotting in physical mapping
EcoRI cuts
BamHI cuts
Cut with one enzyme and electrophorese Blot with
fragments cut with another enzyme ?shows overlap
between bands