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Enzymes for manipulating DNA

*** Buffers and solution conditions***


I. DNA polymerases
III. Kinase and alkaline phosphatase
IV. Nucleases
V. Topoisomerase
Course Readings: 19 and 20

Buffers are crucial for activity of enzymes!


Ideal biochemical buffers:
pKa between 6 and 8
Chemically inert
Polar (soluble and not membrane permeable)
Non-toxic
Inexpensive
Salt and temperature indifferent
Tris: pKa is 8.0
Tris(hydroxymethyl)aminomethane (THAM): the free
base for (pH 7.5-8.5)
Tris-HCl: the acidic form (for pH 7-8)

Tris is widely used, but it isnt perfect:


Buffering is weak below pH 7.5 and above pH 9.0
pH must be measured using a special pH meter
electrode
Toxic to many types of mammalian cell cultures
Tris solution pH changes with temperature! Drops
0.03 pH units for each degree C increase
Tris solution pH changes with concentration!
Example: 10mM Tris pH 7.9, 100mM Tris pH 8.0
Below pH 7.5, use a Good buffer: HEPES, Tricine,
BES, MOPS, MES

Enzyme reaction buffers:


Buffer: Tris, HEPES, etc.
Salt: NaCl, KCl, PO4-, etc.--stabilizes protein
structure, facilitates protein-DNA interactions
Divalent metal ions: Mg2+, Ca2+, Zn2+, etc.-often required for enzyme activity
Glycerol: (for storage)--stabilizes protein
structure
EDTA: chelates (removes) divalent cations-important especially for storage, if your enzyme
is especially sensitive to metal ion-dependent
proteases
Beta mercaptoethanol or dithiothreitol:
reducing agents that prevent illegitimate
disulfide bond formation
Non-specific
protein: Bovine serum albumin

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

A way of making blunt


ended DNA (repair after
fragmentation)
mechanical

A way of radiolabeling DNA

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)

Priming reverse transcriptase:


1) General RNA amplification:
Oligo(dT)12-18
Random sequence oligonucleotides
2) Specific mRNA
Single oligonucleotide sequence
complementary to your mRNA
NOTE: Reverse transcriptase is error-prone
(about 1/500 bp is mutated)

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

Cloning with topoisomerase

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)

Enzymes for manipulating DNA


*** Buffers and solution conditions***
I. DNA polymerases
III. Kinase and alkaline phosphatase
IV. Nucleases
V. Topoisomerase
Course Readings: 19 and 20

Cutting and pasting DNA


I.

Restriction and modification


systems
II. Recognition and cleavage of
DNA by restriction
endonucleases (REases)
III. Joining (ligating) DNA molecules
IV. Cloning techniques

Discovery of
restriction/modification
EOP = efficiency of
plating (a measure of
phage virulence)
= bacteriophage

E. coli K has R/M system


E. coli C has no M system

Cautions for cloning in E.coli


Strains with methylases (dam or dcm)
produce methylated DNA--difficult to cleave
with certain enzymes, hard to transform
some strains
Strains with restriction systems intact will
restrict DNA coming from a host lacking
methylases, or from a host with specific
types of methylations
Best bet is to delete the restriction
systems, but not all cloning strains have this

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

Mode of action of type II REases


EcoRI

5...G^AATTC...3
3...CTTAA^G...5
EcoRI

5...G^35AATTC...3
3...CTTAA53^G...5

Example recognition sequences for


REases

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)

How often does REase cut my


sequence?
1) Known sequence: scan for sites by computer (eg.
at www.rebase.neb.com)
2) Unknown sequence: hypothetical calculations
4 cutter: site occurs randomly every 4 4 (256) base
pairs
6 cutter: every 46 (4096) bp
8 cutter: every 48 (65536) bp
But sequences are not distributed randomly (table
3.4)
3) Sequence context effects
Some sites are preferred over others by enzyme

The ligation reaction

Biological function
of ligases:
Lagging strand
DNA synthesis
genetic
recombination
DNA repair

Behavior of cohesive ends


(overhangs)

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

Creating a recombinant DNA molecule

Plasmid vector:
a cloning vehicle
it can replicate
itself in a bacterial
host and contains
a means for
selection (eg.
antibiotic
resistance)

Ligation efficiency depends on the


DNA ends in the reaction
Complementary sticky ends
Ligation is efficient
annealing of complementary overhangs brings
5P and 3OH into close proximity
Blunt ends
Ligation is inefficient
need high concentrations of ligase and DNA
molecular crowding reagents (like PEG 8000)
improve intermolecular ligation, then dilute to
promote intramolecular ligation
Follow the manufacturers instructions

Cloning foreign DNA by adding linkers

(your DNA
molecule
should not have
EcoRI sites in
this case)

Cloning foreign DNA by adding


adaptors

The advantage
of this is you do
not need to
treat the
adaptormodified DNA
with restriction
enzyme

Terminal transferase to add


polynucleotide tails to foreign DNA and
vector DNA
Foreign
DNA

Vector
DNA
dTTP

Cloning Taq PCR products


Taq PCR products have a 3 A overhang
Prepare vector to have a 3 T overhang
HphI leaves T overhangs

Directional cloning

Directional cloning

This guarantees
the orientation of your DNA fragmen

Easy cloning: PCR products


Design PCR primers with built
in restriction sites (check
amplified sequence for those
sites first!)

Ready for
directiona
l cloning

Utility of
alkaline
phosphatas
e in ligation

Chances of
getting
recombina
nt product
are
improved

Cutting and pasting DNA


I.

Restriction and modification


systems
II. Recognition and cleavage of
DNA by restriction
endonucleases (REases)
III. Joining (ligating) DNA molecules
IV. Cloning techniques

Mobilizing DNA: vectors for


propagation in E. coli
Plasmids
Bacteriophage
M13
Lambda
Cosmids and BACs

Plasmids and transformation


I.
II.

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

a generic, minimal plasmid

restriction
site for
cloning

antibiotic
resistance
pBi430/530
1500 base pairs
(a manageable size

origin of
replicatio
n

Replicon -- how the plasmid replicates


Governs replication of plasmid and number of
plasmid copies per cell (copy number)
A replicon includes:
origin of replication (ori: a site on the DNA)
associated factors

> 30 different replicons known, but most


plasmids used today have pMB1 (or the close
relative colE1) replicon

pMB1/colE1
replication
mechanism
1
Deletion of Rop
or mutation of
RNA II cause
increases in
replication and
copy number

Common plasmids and their stats


PLASMID

REPLICON

COPY #

pBR322

pMB1

15-20

pUC

Modified form 500-700


of pMB1 (RNAII
mutation)

pACYC

p15A

18-22

pSC101

pSC101

about 5

Plasmid copy number


High copy number plasmids
Workhorses of molecular cloning
Used for almost all routine manipulation of
small (<15 kb) recombinant DNAs
Low copy number plasmids
For genes that are lethal or unstable in high
copy number plasmids
For constructing Bacterial Artificial
Chromosomes (BACs) that can propagate
large (>100 kb) recombinant DNAs

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

Antibiotic resistance genes


Beta lactamase (bla): breaks down
ampicillin and carbenicillin (inhibitors of
cell wall synthesis). Cells carrying this
gene are often termed ampr
CAUTION: Over time beta-lactamase is
secreted into the medium where it breaks
down the antibiotic and depletes it.
Eventually this allows the growth of
ampicillin/ carbenicillin sensitive cells,
defeating the selection

Antibiotic resistance genes


Chloramphenicol acetyl transferase (CAT):
inactivates chloramphenicol (cm), which
normally inhibits peptidyl transferase activity of
the ribosome (no protein synthesis = dead cell)
Another use for cm:
replication of plasmids with pMB1/colE1
replicons is not inhibited by cm
Cm-treated cells stop growing but continue
making these plasmids, this is a way to
amplify plasmid copy numbers prior to a
plasmid prep

Antibiotic resistance genes


Tet A (C ) protein: confers resistance to tetracycline (an
inhibitor of protein synthesis) by pumping this antibiotic
out of the cell
Bacterial aminophosphotransferases: confer resistant to
kanamycins (aminoglycoside antibiotics that inhibit
protein synthesis) by transferring the gamma phosphate
of ATP to a 3 hydroxyl group of the kanamycin

The ideal plasmid


1. Confers a readily selectable phenotypic
trait
2. Has single sites for many restriction
enzymes
3. Low molecular weight
-- Gives higher copy #, stability, and
transforming efficiency
-- Can accept larger pieces of DNA
-- Easier to handle (less susceptible to
breakage)

pBR322
The first widely useful cloning vehicle

Created using
transposition and
restriction/ligation
reactions

Utility of pBR322:

pBR322

Clone into sites in the


Tcr gene, which allows
identification of
recombinants--these
will be amp resistant
but tet sensitive
(initially plate on
ampicillin, then
replica plate on
tetracycline plates).
But: pBR322 has low
copy number, large
size, and too few
options for cloning
sites

Boldface indicates the restriction site


is present in only one site within the
plasmid

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

Host strain encodes the Cterminus of beta galactosidase


(omega fragment)
Beta galactosidase function is
only seen in the presence of
both the N- and C-terminal
fragments
Beta gal function can be
monitored by the cleavage of
X-gal which yields a bright blue
product (blue colonies
on a

Bright blue

An alpha complementing plasmid vector


(MCS)

pUC 19

(no Beta galactosida


DNA in the MCS interrupts the lacZ gene

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

Third generation cloning vectors:


specialized plasmids
Vectors containing bacteriophage RNA polymerase
promoters: for production of a specific RNA (probe
synthesis, in vitro translation, etc.)
Low copy number vectors: for cloning of unstable or
toxic genes
Vectors designed for expression of specific proteins
(for further purification and biochemical
characterization). Proteins may be synthesized with
tags to assist in purification

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,

on ice for 30 min.


DNA uptake by cells Heat shock (42C) for 1.5
minutes
Cells recover
Add growth media, 37C for 1
hour
Selection occurs
Plate on growth medium plus
selection (antibiotic) for the
plasmid
If cells are good: Efficiency ~ 106 - 107
cells/microgram
plasmid DNA

Ultra competent cells (chemical)


5 x 108 transformants/microgram plasmid
See protocol 23 of Molecular Cloning ch. 1
Treat with
MnCl2

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)

When cloning a piece of DNA consider:


1) Choice of vector: what kind of plasmid vector to
use (which restriction sites can be used in the
vector)?
2) Ligating DNA to vector: how will the ligation
reaction be set up to facilitate getting what you
want?
3) Moving DNA by transformation: what strain of E.
coli will you transform into? Which method for
transformation?
4) Screening for successful ligation products
(recombinant plasmid DNA): how will the
plasmids be identified?

recombinant

Setting up a transformation-how will the competent cells be


treated?
1. No plasmid (negative control, nothing
should grow on this plate)
2. Supercoiled plasmid of a known
concentration (to determine efficiency of
competent cells)
3. Vector DNA (dephosphorylated?) ligated
without insert DNA (background
transformants)
4. Vector DNA ligated with insert DNA
(desired products)

Example outcome of a successful


transformation: chemically competent
cells
1) No DNA--No colonies
2) 2 nanograms (10-9 g, 10-3 micrograms)
supercoiled plasmid DNA--500 colonies
(efficiency of cells: 2.5 x 105 transformants
per microgram DNA)
3) Vector alone--small number of colonies
4) Vector plus insert--larger number of colonies
than for #3

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

Mobilizing DNA: vectors for


propagation in E. coli
Plasmids
Bacteriophage
M13
Lambda
Cosmids and BACs