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DNA polymerases--making
copies, adding labels, or fixing
DNA
E. coli DNA polymerase I --the classic DNA
polymerase
Moderately processive polymerase
3'->5' proof-reading exonuclease
5'->3' strand-displacing (nicktranslating) exonuclease
Used mostly for labelling DNA
molecules by nick translation. For other
purposes, the Klenow fragment is
usually preferred
DNA polymerases
Klenow fragment --the C-terminal 70% of E.
coli DNA polymerase I; originally prepared as
a proteolytic fragment (discovered by
Klenow); now cloned
Lacks the 5'->3' exonuclease activity
Uses include:
Labeling DNA termini by filling in the
cohesive ends generated by certain
restriction enzymes
generation of blunt ends
DNA sequencing
DNA polymerases
Native T7 DNA polymerase --highly processive,
with highly active 3'->5' exonuclease
Useful for extensive DNA synthesis on long,
single-stranded (e.g. M13) templates
Useful for labeling DNA termini and for
converting protruding ends to blunt ends
Modified T7 polymerase (Sequenase) --lack of
both 3'->5' exonuclease and 5'->3' exonuclease
Ideal for sequencing, due to high processivity
Efficiently incorporates dNTPs at low
concentrations, making it ideal for labeling
DNA
DNA polymerases
Reverse transcriptase
RNA-dependent DNA polymerase
Essential for making cDNA copies of
RNA transcripts
Cloning intron-less genes
Quantitation of RNA
Reverse transcriptase:
The Km for dNTPs is very high (relatively nonprocessive)
Makes a DNA copy of RNA or DNA
-- but -The self-primed second strand synthesis is
inefficient
Second-strand cDNA synthesis is usually done
with DNA polymerase and a primer
How RT works
cDNA library
construction
using
reverse
transcriptas
e
cDNA Library
Construction Kit
(Clontech)
Terminal transferase
template-independent DNA polymerase
Incorporates dNTPs onto the 3' ends of
DNA chains
Useful for adding homopolymeric tails or
single nucleotides (can be labelled) to the
3' ends of DNA strands (make DNA
fragments more easily clonable)
T4 polynucleotide kinase
Transfers gamma phosphate of ATP to
the 5 end of polynucleotides
Useful for preparing DNA fragments for
ligation (if they lack 5 phosphates)
Useful for radiolabelling DNA fragments
using gamma 32P ATP as a phosphate
donor
alkaline phosphatase
Catalyzes removal of 5 (and 3)
phosphates from polynucleotides
Useful for treating restricted vector DNA
sequences prior to ligation reactions,
prevents religation of vector in the
absence of insert DNA
Lack of vector 5 phosphates may inhibit
transformation efficiency? Use only when
absolutely necessary
Nucleases
Exonucleases
Remove nucleotides one at a time from a
DNA molecule
Endonucleases
Break phosphodiester bonds within a DNA
molecule
Include restriction enzymes
Exonucleases
Bal 31
Double-stranded exonuclease,
operates in a time-dependent manner
Degrades both 5 and 3 ends of DNA
Useful for generating deletion sets,
get bigger deletions with longer
incubations
Exonucleases
Exonuclease III--double-stranded DNA
3-5 exonuclease activity
3 overhangs resistant to activity, can
use this property to generate
nested deletions from one end of a
piece of DNA (use S1 nuclease to
degrade other strand of DNA)
Exonucleases
Exonuclease I
3-5 exonuclease
Works only on single-stranded DNA
Useful for removing unextended
primers from PCR reactions or other
primer extension reactions
Endonucleases
Dnase I
Cleaves double-stranded DNA
randomly (also cleaves singlestranded DNA)
Mn++: both strands of DNA cut
Mg++: single strands nicked
Very useful for defining binding sites
for DNA binding proteins
DNAse I
footprinting
Calibrate
the nicking:
1 hit per
DNA
molecule
DNAse I
footprinting
:
Drosophila heat-shoc
factor
Gel
following
footprinting
reaction
Sites for
interaction of
HSF with DNA
Topoisomerase
Function:
A restriction enzyme and ligase--all in one
altering the linking number in coiled, constrained
(supercoiled) DNA--relaxing DNA twisting during
replication
Model for function:
http://mcb.berkeley.edu/labs/berger/structures.html#modeling
Topoisomerase
Topoisomerase catalyzed ligation is
EXTREMELY efficient (>85% of resulting
plasmids are recombinant)--excellent for
library constructions
Can be used to clone blunt ended DNA (PCR
products, restriction digests), T-overhang PCR
products (from Taq polymerase), and
directional clones
You have to use their plasmid vectors (ie.
forget about using your favorite lab plasmid
unless you know how to covalently attach
topoisomerase)
Discovery of
restriction/modification
EOP = efficiency of
plating (a measure of
phage virulence)
= bacteriophage
deletion
Types of endonucleases
Type I: multisubunit proteins that function as a single protein
complex, usually contain two R subunits,two M subunits and one
S subunit
Type II: recognize specific DNA sequences and cleave at constant
positions at or close to that sequence to produce 5-phosphates
and 3-hydroxyls. Most useful in cloning!!
Type III: composed of two genes (mod and res) encoding protein
subunits that function either in DNA recognition and modification
(Mod) or restriction (Res)
Type IV: one or two genes encoding proteins that cleave only
modified DNA, including methylated, hydroxymethylated and
glucosyl-hydroxymethylated bases
5...G^AATTC...3
3...CTTAA^G...5
EcoRI
5...G^35AATTC...3
3...CTTAA53^G...5
4cutters:
AluI
5...AG^CT...3
bluntends
MspI
5...C^CGG...3
5overhang(2bp)
PvuII
5...CAG^CTG...3
bluntends
KpnI
5...GGTAC^C...3
3overhang(4bp)
5...GC^GGCCGC...3
5overhang(4bp)
6cutters
8cutters
NotI
Unusualsites
MwoI
5...GCNNNNN^NNGC...3
3...CGNN^NNNNNCG...5
3overhang
(3bp)
Biological function
of ligases:
Lagging strand
DNA synthesis
genetic
recombination
DNA repair
Cloning techniques
A) Modify the ends of the DNAs to make
foreign DNA sequences more ligate-able
B) Directional cloning (generate easily
cloned PCR fragments)
C) Treat the vector DNA with alkaline
phosphatase to improve the efficiency
of ligation of foreign DNA versus vector
recircularization
Plasmid vector:
a cloning vehicle
it can replicate
itself in a bacterial
host and contains
a means for
selection (eg.
antibiotic
resistance)
(your DNA
molecule
should not have
EcoRI sites in
this case)
The advantage
of this is you do
not need to
treat the
adaptormodified DNA
with restriction
enzyme
Vector
DNA
dTTP
Directional cloning
Directional cloning
This guarantees
the orientation of your DNA fragmen
Ready for
directiona
l cloning
Utility of
alkaline
phosphatas
e in ligation
Chances of
getting
recombina
nt product
are
improved
Properties of plasmids
Plasmids as cloning vehicles
(vectors)
III. Ligation and transformation, and
identification of recombinant
plasmids
Course Readings: #21 (plasmids) and
#22 (antibiotic selection)
Plasmids
Extrachromosomal, double-stranded, usually
circular, supercoiled DNA molecules
Found in many bacterial species
Replicate and are inherited independently of
the bacterial chromosome
Maintain copy number in cell through an
origin of replication (replicon)
Usually have genes coding for enzymes that
provide benefits for the host bacterium, eg.
antibiotic resistance
restriction
site for
cloning
antibiotic
resistance
pBi430/530
1500 base pairs
(a manageable size
origin of
replicatio
n
pMB1/colE1
replication
mechanism
1
Deletion of Rop
or mutation of
RNA II cause
increases in
replication and
copy number
REPLICON
COPY #
pBR322
pMB1
15-20
pUC
pACYC
p15A
18-22
pSC101
pSC101
about 5
Plasmid maintenance
Plasmids contain selectable markers: genes
carried by the plasmid that confer functions
required for host survival
Selection: only those cells with the plasmid
will survive
Allows transformation (a rare event) to be
feasible
A way to keep cells from losing plasmids
that may otherwise confer a selective
disadvantage
pBR322
The first widely useful cloning vehicle
Created using
transposition and
restriction/ligation
reactions
Utility of pBR322:
pBR322
pUC plasmids
second generation cloning vectors
Reduced size (about 2000 bp)
Multiple cloning site (MCS, also called polylinker): unique sites for lots of different
restriction enzymes
Very high copy number (mutation in RNA II)
New blue-white screening tool for
recombinants (alpha complementation is
disrupted by foreign DNA in the MCS)
Alpha complementation
Plasmid encodes N-terminus
of beta galactosidase (alpha
fragment)
X-gal
Bright blue
pUC 19
Alpha complementation
Plasmid encodes N-terminus of beta
galactosidase (alpha fragment), with an MCS
Foreign DNA in the MCS, no alpha fragment
No alpha fragment, no B-gal
No B-gal, no blue color (white colonies)
Colony without
foreign DNA in
MCS
pUC19
transformation
plate
Colony with
foreign DNA in
Transformation of E.coli
with plasmid DNA
E.coli strain: must be antibiotic sensitive, best if
it lacks restriction-modification systems
Make cells take up DNA by
Chemical competence
Electroporation
(natural competence--not E.coli though)
Chemically competent
cells-basic method
Grow cells to A600 of 0.4, spin to get cell
pellet
Resuspend cells in CaCl2 (100 mM), pellet
again
Resuspend in small volume of
CaCl2/glycerol
Freeze cells (-80C) or go straight to
transformation protocol
Transformation of chemically
competent cells
DNA binds to cells Mix DNA and competent cells,
CaCl2
KCl
Hexammine CoCl2
Store in DMSO
(protocol rather difficult, inconsistent)
These can be bought
Transformation by electroporation
> 109 transformants/microgram DNA (ideally)
Grow cells to A600 of 0.4
Centrifuge and resuspend in water + 10% glycerol
(do this 4 times to reduce conductivity)
Place cells with DNA in electrode-containing
cuvette, deliver electrical pulse
If there is arcing (sparks) transformation efficiency
will be poor (uneven transfer of charge). To avoid
this make sure the ion concentration is very low
(less than 10 mM salt)
recombinant
Identifying recombinant
plasmid-containing cells
Alpha complementation: most white colonies
represent presence of insert DNA blocking
functional beta galactosidase
Increase in number of transformants in
presence of insert vs. absence of insert
Insert treated with alkaline phosphatase
Directional cloning--preventing religation of
vector
Must screen colonies/plasmids for inserts,
usually by PCR
Confirm clones
by sequencing