Enzymes for manipulating DNA

*** Buffers and solution conditions***

I. DNA polymerasesIII. Kinase and alkaline phosphataseIV. NucleasesV. 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

• 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

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,

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 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

0DNAse I

footprinting:

Gel following

footprinting reaction

Drosophila heat-shockfactor

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 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)

Topoisomerase

Enzymes for manipulating DNA

*** Buffers and solution conditions***

I. DNA polymerasesIII. Kinase and alkaline phosphataseIV. NucleasesV. 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 moleculesIV. Cloning techniques

Discovery of restriction/modification

EOP = efficiency of plating (a measure of phage virulence)

= bacteriophage

E. coli K has R/M systemE. 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

5´ ... G^A A T T C ... 3´3´ ... C T T A A^G ... 5´

5´ ... G^ 3’ 5’ A A T T C ... 3´3´ ... C T T A A 5’ 3’ ^G ... 5´

EcoRI

EcoRI

Example recognition sequences for REases

4-cutters:

AluI 5´ ... AG^CT ... 3´ blunt ends

MspI 5´ ... C^CGG ... 3´ 5’ overhang (2 bp)

6-cutters

PvuII 5´ ... CAG^CTG ... 3´ blunt ends

KpnI 5´ ... GGTAC^C ... 3´ 3’ overhang (4 bp)

8-cutters

NotI 5´ ... GC^GGCCGC ... 3´ 5’ overhang (4 bp)

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 calculations4 cutter: site occurs randomly every 44 (256) base

pairs6 cutter: every 46 (4096) bp8 cutter: every 48 (65536) bpBut sequences are not distributed randomly (table

3.4)3) Sequence context effects

Some sites are preferred over others by enzyme

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

The ligation reaction

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

dTTP

Foreign DNA

Vector DNA

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

Ready for directional cloning

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

Utility of alkaline

phosphatase in ligation

Chances of getting recombinant 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 moleculesIV. Cloning techniques

Mobilizing DNA: vectors for propagation in E. coli

•Plasmids•Bacteriophage

M13 Lambda

•Cosmids and BACs

Plasmids and transformation

I. Properties of plasmidsII. 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

origin of replication

antibiotic resistance

restriction site for cloning

pBi430/5301500 base pairs

(a manageable size

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

1

2

3

4

pMB1/colE1 replication mechanism

Deletion of Rop or mutation of RNA II cause increases in replication and copy number

PLASMID REPLICON COPY #

pBR322 pMB1 15-20

pUC Modified form of pMB1 (RNAII mutation)

500-700

pACYC p15A 18-22

pSC101 pSC101 about 5

Common plasmids and their stats

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

pBR322

Utility of 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 plasmidssecond 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)

• Host strain encodes the C-terminus 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

X-gal

pUC 19

An alpha complementing plasmid vector

DNA in the MCS interrupts the lacZ gene (no Beta galactosidase)

(MCS)

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)

pUC19 transformation plate

Colony without foreign DNA in MCS

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 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 (-80°C) or go straight to transformation protocol

Transformation of chemically competent cells

• Mix DNA and competent cells, on ice for 30 min.

• Heat shock (42°C) for 1.5 minutes

• Add growth media, 37°C for 1 hour

• Plate on growth medium plus selection (antibiotic) for the plasmid

Efficiency ~ 106 - 107 cells/microgram plasmid DNA

DNA binds to cells

DNA uptake by cells

Cells recover

Selection occurs

If cells are good:

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) No DNA--No colonies2) 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 colonies4) 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