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 isn’t 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 (BSA)
•Other cofactors, eg. ATP, NADH
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 (nick-translating)
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 mechanical
fragmentation)
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, singlestranded (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 non-processive)
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
transcriptase
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 single-stranded 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:
Gel following
footprinting
reaction
0
Drosophila heat-shock
factor
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.
II.
Restriction and modification systems
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 glucosylhydroxymethylated bases
Mode of action of type II REases
EcoRI
5´ ... G^A A T T C ... 3´
3´ ... C T T A A^G ... 5´
EcoRI
5´ ... G^ 3’
5’ A A T T C ... 3´
3´ ... C T T A A 5’
3’ ^G ... 5´
Example recognition sequences for REases
4-cutters:
AluI
5´ ... AG^CT ... 3´
blunt ends
MspI
5´ ... C^CGG ... 3´
5’ overhang (2 bp)
PvuII
5´ ... CAG^CTG ... 3´
blunt ends
KpnI
5´ ... GGTAC^C ... 3´
3’ overhang (4 bp)
5´ ... GC^GGCCGC ... 3´
5’ overhang (4 bp)
6-cutters
8-cutters
NotI
Unusual sites
MwoI
5´ ... GCNNNNN^NNGC ... 3´
3’ overhang
3´ ... CGNN^NNNNNCG ... 5´
(3 bp)
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 44 (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 5’P and
3’OH 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 manufacturer’s 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
adaptor-modified
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 fragment
Easy cloning: PCR products
Design PCR primers with built in
restriction sites (check amplified
sequence for those sites first!)
Ready for
directional
cloning
Utility of
alkaline
phosphatase
in ligation
Chances of
getting
recombinant
product are
improved
Cutting and pasting DNA
I.
II.
Restriction and modification systems
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.
III.
Properties of plasmids
Plasmids as cloning vehicles (‘vectors’)
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
replication
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
2
3
4
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
500-700
pACYC
Modified form of
pMB1 (RNAII
mutation)
p15A
pSC101
pSC101
about 5
18-22
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 “poly-linker”):
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 plate)
Bright blue
An alpha complementing plasmid vector
(MCS)
pUC 19
DNA in the MCS interrupts the lacZ gene (no Beta galactosidase)
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 MCS
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 cellsbasic 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 (-80°C) or go straight to
transformation protocol
Transformation of chemically
competent cells
• Mix DNA and competent cells, on
ice for 30 min.
DNA uptake by cells
• Heat shock (42°C) for 1.5 minutes
• Add growth media, 37°C for 1
Cells recover
hour
• Plate on growth medium plus
Selection occurs
selection (antibiotic) for the
plasmid
DNA binds to cells
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 recombinant plasmids be
identified?
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)
2)
3)
4)
No DNA--No colonies
2 nanograms (10-9 g, 10-3 micrograms) supercoiled
plasmid DNA--500 colonies (efficiency of cells: 2.5
x 105 transformants per microgram DNA)
Vector alone--small number of colonies
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