PCR- Polymerase chain reaction PCR is an in vitro technique for the amplification of a region of DNA which lies between two regions of known sequence. PCR amplification is achieved by using oligonucleotide primers. These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence. The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. This results in the synthesis of new DNA strands which are complementary to the parent template strands. These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers) The oligonucleotide directed synthesis of daughter DNA strands can be repeated if the new duplex is denatured (by heating) and additional primers are allowed to anneal (by cooling to an appropriate temperature).

The steps of the PCR reaction: Template denaturation Primer annealing Primer extension These steps comprise a single "cycle" in the PCR amplification methodology. After each cycle the newly synthesized DNA strands can serve as templates in the next cycle.

Optimization of PCR a) Mg++ - one of the main variables – affects stability of double helix, used to mimic temperature b) Template DNA concentration – one template strand of DNA is needed. to reduce error by Taq DNA polymerase, a higher DNA concentration can be used Too much template may increase the amount of contaminants and reduce efficiency. c) Enzymes used – choose the appropriate one for the length or degree of fidelity required d) dNTP - can use up to 1.5 mM dNTP, want in excess. dNTP chelate Mg++. Excessive dNTP can increase the error rate and possibly inhibits Taq. Lowering the dNTP (10-50 uM) may reduce error rate Larger size PCR fragment need more dNTP. e) primers - up to 3 uM of primers may be used, but high primer to template ratio can results in non-specific amplification and primer-dimer formation

PCR optimization continued f) Thermal cycling denaturation time can be increased if template GC content is high (calculate Tm). Extension time should be extended for larger PCR products. Extension time is also affected by the enzymes used e.g for Taq - assume 1000 base/min The number of cycles can be increased if the number of template DNA molecules is very low, and decreased if high amount of template DNA is used g) Additives - Glycerol (5-10%), formamide (1-5%) or DMSO (2-10%) can be added in PCR for template DNA with high GC content (they change the Tm of primer-template hybridisation reaction and the thermostability of polymerase enzyme). 0.5 -2M Betaine (stock solution - 5M) is also useful for PCR over high GC content and long stretches of DNA (Long PCR ) Betaine is often the secret (and unnecessarily expensive) ingredient of many commercial kits. BSA (up to 0.8 µg/µl) can also improve efficiency of PCR reaction. h) PCR buffer Higher concentration of PCR buffer may be used to improve efficiency. Wilsons buffer may work better than the buffer supplied from commercial sources especially with hard to amplify pieces of DNA

In designing primers for PCR,consider:: PCR optimization continued i) Primer design In designing primers for PCR,consider:: length of individual primers between 18-24 bases. Minimum of 15 to be specific it is desirable that the two primers have a close melting temperature or Tm (say, within 5 o C or so). if possible, primer sequence should end with 1-2 GC pairs (GC clamp) each primer pair should be tested for primer-primer interactions. primer sequences should be aligned with all DNA sequences entered in the databases (using BLAST programs) and checked for similarities with repetitive sequences or with other loci, elsewhere in the genome. cycling conditions and buffer concentrations should be adjusted for each primer pair, so that amplification of the desired locus is specific, with no secondary products

http://frodo.wi.mit.edu/- Primer3 Primer3 is a widely used program for designing PCR primers. Primer3 can also design hybridization probes and sequencing primers. PCR is used for many different goals. Consequently, primer3 has many different input parameters that you control and that tell primer3 exactly what characteristics make good primers for your goals.

PCR methods Hot-start PCR- preparing PCR at room temperature can generate secondary non specific products in the first PCR cycle that are amplified in subsequent cycles. Hot PCR prevents non-specific extension at ambient temperatures by either excluding or reversibly inhibiting the polymerase enzyme. Most common way to do this is separate the polymerase with wax until after the first denaturing step. "Touch-down" PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. An annealing temperature that is higher than the target optimum is used in early PCR cycles. The annealing temperature is decreased by 1°C every cycle or every second cycle until a specified or 'touchdown' annealing temperature is reached. The touchdown temperature is then used for the remaining number of cycles. This allows for the enrichment of the correct product over any non-specific product.

PCR methods cont. Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers. Reduces the contaminations in products due to the amplification of unexpected primer binding sites. Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested, the desired fragment is circularized by ligation, then PCR using primer complementary to the known sequence extending outwards. (see next slide) AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).

Inverse PCR

PCR methods cont. RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. RACE (rapid amplificaton of cDNA ends) - used where information about DNA/protein sequence is limited. It allows amplification of an unknown end portion of a transcript using known information from the centre. It can be used to amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA.

Mutagenesis by PCR – used to introduce mutations using the primers PCR methods cont. Multiplex-PCR - 2 or more unique targets of DNA sequences in the same specimen are amplified simultaneously. E.g. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens. Asymmetric PCR –results in synthesis of mainly ssDNA by the most abundant primer. Useful for making probes or primers. In Situ PCR – done on a slide to identify where in a section or cell a certain transcript or piece of DNA is located Mutagenesis by PCR – used to introduce mutations using the primers