Biology 224Instructor: Tom Peavy

March 20, 2008

<Figures from PCR by McPherson & Moller>

PolymeraseChain

Reaction

PCR= Polymerase Chain Reaction

• “DNA photocopier”• integral tool for molecular biologists• work horse• versatile (many applications)• not difficult to perform technically • fast

• Kary Mullis of Cetus Corp invented PCR in 1983 (Klenow fragment of DNA pol I)• First paper describing the use of Taq polymerase was in 1988 (Saiki et al., 1988)• PCR patent issues involving Taq polymerase

PCR applications

• Cloning cDNAs and RAPD• Cloning genes• Real-time PCR• PCR mutagenesis • PCR probe generation for hybridization• Population sampling and genotyping • Genomic fingerprinting RAPD-PCR multiplex-PCR PCR-VNTRs Micro- and Minisatelline repeat-PCR • Diagnostic PCR (detection of pathogens, GMOs, etc.)

PCR components

Template DNAPrimers

dNTPs (water, buffer)

Thermostablepolymerase

1) Template DNA is denatured (Denaturation phase; 94C)

2) Primers allowed to anneal to template; Tm of primers is important (Annealing phase; variable temperature)

3) Increase temperature to optimum for thermostable polymerase (Elongation phase; 68-72C)

4) Repeat the whole cycle starting at step 1

PCR Kinetics

Early cycles: primers act like probes searching for complementary Sequences on template DNA

Mid cycles: amplication process isis fully underway with an exponentialaccumulation of amplimers

Late cycles: reagents are limiting andAmplification is suboptimal

Sources of Template DNA

Genomic DNA

RNA isolation and cDNA

Plasmid, bacteriophage, cosmid and artifical chromosome DNA

Pathological and forensic samples

Archaeological samples

Template amount

-very sensitive technique (don’t need much target template)but the amount of template is likely to require optimization(generally <1 nanogram of cloned template and up to amicrogram of genomic DNA are used)

-relative amount of target can be increased (e.g. choose cDNA librarywhere target should be expressed in large amounts)

Technical Difficulties

Mispriming – primers anneal to alternate sites and not to “correct” or targeted site

Needle-in-a-haystack (Template in limited amounts)

Mismatches allowed internally if annealing temperatureis low (below Tm)

Misprimed PCR products will continue to be amplified (PCR primers are incorporated into the amplimer at the

terminal end and will thus serve as a perfect match for futurePCR cycles; large amounts of PCR product accumulateif in it occurs in the early cycles)

PCR of non-specific sequences or misprimed products leads to either smeary gels or unexpected amplimers sizes

Artifactual products on agarose gels can arise from Primer-Dimer formation

Contamination Problems

Carry-over contaminationprior PCR products, clones, or samples with DNA in general(e.g. cell lysates, genomic or plasmid DNA preparations, etc)can enter the PCR tube and serve as potential template

-most often due to aerosol from pipetteman

Other template contamination can be due to:- floating debris (circulation/vents)-laboratory surfaces-tissue from self or others (e.g. skin, hair)-solution contamination

Preventative: aerosol-free tips (cotton plugs), UV irradiation,Designated PCR set-up area, gloves, premixes, aliquots of reagents

Optimization of PCR

To improve specificity:template qualityoptimize concentrations of Mg2+, other ions, primers,

dNTPs and polymeraseefficient denaturation, high annealing temps, and fast ramping rateslimiting number of cycles and their lengthPCR strategies (e.g. touchdown PCR, hot start PCRnested PCR)Primer design

-Magnesium ion concentration often needs optimization exists as dNTP-Mg2+ complexes, interacts with DNA backbone influences activity of Taq polymerase;

MgCl2 is used in buffer to adjust concentrations

Between 0.5 and 5mM is generally used (1.5 mM common)Low concentrations tend to have low yields of PCR product High concentrations tend to reduce the fidelity of Taq polymerase and lead to amplification of non-specific products

NaCl or KCl concentrations also can be optimized

Magnesium ions are critical

To enhance for efficient denaturation, high annealing temps, and fast ramping rates:

Use quality PCR machines (may not effectively reach temperatures or takes long time to ramp)

Use thin walled PCR tubes

Use Polymerase with High fidelityTaq polymerase (from thermophilic bacterium Thermus aquaticus) = 94 kDa protein with 2 catalytic properties

1. 5’3’ DNA polymerase (elongates 50-60 nucleotides/second)2. 5’3’ exonuclease (removes nts in front of growing strand)

Recombinant versions of Taq (enhanced for either purification or performance)

Lacks 3’5’ exonuclease activity

Fidelity of Taq or error rate is: 1 base misincorporation per 104 nucleotides polymerizedfor a 400 bp fragment amplied 106 fold (=20 cycles) results inin about 33% of the products carrying a mutation(thus should sequence several PCR amplimers to determineconsensus)

Proofreading DNA polymerases (= those that contain 3’5’ exonuclease activity)

Proofreading ability is due to the capacity of the enzyme to discriminatebetween whether the nucleotide at the 3’ OH of an extendingstrand is correctly or incorrectly paired with the template strand

Generally these enzymes are even more thermostable and tolerant ofbuffer conditions

However, could chew up mismatched 3’ primer ends also (‘nibbling’)

Increases fidelity about 5-12 depending on enzyme

Examples: Vent®, DeepVent®, Tli (Thermoccocus litoralis), Pfu (Pyrococcus furiosus), 12 fold

Usually leaves blunt ends for cloning rather than overhangs

Hot Start PCR Used to overcome non-specific annealing of primers and/or

primer-dimer formation prior to the denaturation step(annealed primers will be extended as temperatureramps up to denaturation temp)

Cheapest method is to add polymerase after temperature isis above 70C

Alternatively can use commercial reagents such as Taq that hasan antibody attached so as to prevent polymerase activityuntil the antibody is denatured (> 70C)

Touchdown PCR

Used to increase specific PCR productsAnnealing temperature is set slightly above the Tm of the primers in the early cycles (enhances the chances of specific annealing of primers vs. non-specific)

Annealing temperature is gradually lowered in subsequent cycles (e.g. 1C every two cycles) until desired lower limiting annealing temperature is reached

Effect is that the target sequences are preferentially amplified in early cycles and then are continued to be amplified exponentially (out competing non-specific targets)

Nested PCR

Design two outside primers for thefirst reaction,

Then use a portion of the first reaction as template in a second reaction usingInternal ‘nested’ primers

Primer length and sequence are of critical importance in designing the parameters of a successful amplification: the melting temperature of a DNA duplex increases both with its length, and with increasing (G+C) content: a simple formula for calculation of the Tm is: Tm = 4(G + C) + 2(A + T)oC

Annealing Temperature and Primer Design

In setting the annealing temperature of PCR reaction:• As a rule of thumb, use an annealing temperature (Ta) about 5oC below the lowest Tm of the pair of primers to be used if a good yield of product is desired• Alternatively, if an increased specificity is desired, one can either Perform touchdown PCR (high-low anneal temp)

The Tm of the two primers should not be different because it may never give appreciable yields of product due to trade-offs (annealing temperature appropriate for one but not the other)

Can result in inadvertent "asymmetric" or single-strand amplification of the most efficiently primed product strand.

Note: Annealing does not take long: most primers will anneal efficiently in 30 sec or less, unless the Ta is too close to the Tm, or unless they are unusually long.

The optimum length of a primer depends upon its (A+T) content, and the Tm of its partner (to avoid large differences)

Another prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low.

Lengths are generally 17-25mers (rationale: there is a ¼ chance of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4,294,967,296, or 4 billion):

Primer Length

Primers can be designed with engineered sites at the 5’end (e.g. restriction enzyme sites, mutations)

Mismatches can also be designed internally to facilitate in situ mutations (change coding sequence or create restriction sites)

EcoRI

Note: only use the annealing portion to calculate Tm

For amplification of sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers:

Degenerate primers= a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences.

Need to examine all the options for particular amino acids withRespect to their codon degeneracy

Degenerate Primers

For the opposite direction (5’ end race)need to reverse complement the sequence!

5’ 3’

CGN CTG TGN CTT ACC CTG TTT CCN CTT GTG CCN A C A C C A

3’ 5’

NCC GTG TTC NCC TTT GTC CCA TTC NGT GTC NGC A C C A C A

5’ 3’

complement

reverse

Design of degenerateprimers based on aminoacid sequencing:

If you do not know wherethe peptide regions arelocated in the gene,then need to design PCR primers in bothdirections and tryvarious combinations

Degeneracies obviously reduce the specificity of the primer(s), meaning mismatch opportunities are greater, and background noise increases

Increased degeneracy means concentration of the individual primers decreases (of which there is only one exact match) thus, greater than 512-fold degeneracy should be avoided.

GTG TTC NCC TTT GTC CCA TTC NGTA C C A C

5’ 3’

(24mer) degeneracy= (1/4)2 (1/2)5 = 1/512

Can use deoxyinosine (dI) at degenerate positions rather than use mixed oligos:

dI base-pairs with any other base, effectively giving a four-fold degeneracy at any postion in the oligo where it is present

This lessens problems to do with depletion of specific single oligos in a highly degenerate mixture, but may result in too high a degeneracy where there are 4 or more dIs in an oligo

- primers should be 17-25 bases in length; - base composition should be 50-60% (G+C); - primers should end (3') in a G or C, or CG or GC (prevents "breathing" of ends and increases efficiency of priming) - Tms between 55-80oC are preferred; - runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability

of annealing), and should be avoided; - 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product; - primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.

General Rules for Primer Design

Examples of inter- and intra-primer complementarity which would result in problems:

Real-time PCR quantitation

- uses multiple PCR primer sets to amplify Two or more products within single reaction

- used for genotyping applications where simultaneous analysis of multiple markers is advantageous (or statistically necessary)

- Can amplify over short tandem repeats (STRs)

Multiplex PCR

Short Tandem Repeats (STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another