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At the end of this lecture, students will be able to: define PCR Describe components required for a PCR reaction and their function Elaborate basics of PCR cycling Tell how to check on PCR product Explain factors affecting PCR reaction Explain uses of PCR and their application in life sciences
(b) -
Template Usually DNA, can be RNA (RT-PCR) Contains sequence to be amplified can come from a variety of sources (genomic DNA, other cloned DNA fragments or libraries of DNA fragments) (c) Primers Must flank the region to be amplified Must anneal to opposite strands of DNA primers can be constructed in the lab, or purchased from commercial suppliers The length (and sequence composition) of the oligonucleotide primer also determines the temperature at which we anneal the denatured template and primers to form specific hybrids which can be extended by the DNA polymerase. Tm = 2oC ( A + T) + 4 oC ( G + C) Usually primer length is 18-30 bp, single stranded most critical component of the PCR reaction -determine the specificity Higher GC content is better (>50% GC) Primers should be GC rich at 3 end Not self complementary No primer dimers
PCR cycle
The basic reaction consists of three steps: Template denaturation - double-stranded DNA template is denatured by heating to 95oC/94oC Annealing - The single stranded template is allowed to hybridize to short single stranded oligonucleotide primers - Annealing temp. affects specificity of binding, at too high a temperature primers will remain dissociated, at too low a temp. there will be mismatch hybrid. - Determination of Tm ( melting temperature) for template/primer hybrid can be calculated using the formula; Tm = (4x [G + C]) + (2 x [A + T]) oC Extension - These oligonucleotides are then recognized by DNA polymerase and the strands are replicated. Extension : ~ 72oC 74oC
Amplifications are carried out in cycles. Denature, anneal and extend-repeat the cycle 30-35 times
Time
PCR cycle
At the end of the cycle, we have doubled the number of DNA strands corresponding to the target sequences downstream of the specific primers The doubling of the number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula Amplification = 2n where n=number of cycles
Has it worked?
Check PCR product by agarose gel electrophoresis Is the product the size you expected? Is there more than one band if yes requires optimisation
PCR
Applications of PCR
To study minute quantities of DNA
forensic analysis genetic fingerprinting archaeology and palaeontology (preserved and fossilised material)
Clinical diagnosis
type mutations rapidly RFLP diagnosis of pathogenic diseases
Applications of PCR
Quantification of mRNA
RT-PCR Measure relative amounts of mRNA in tissues