PCR

Question 1

PCR, function, origin, steps

Answer 1

selectively amplifying a particular segment of DNA

starting material does not have to be highly purified e.g. cell lysate or
colonies can be used directly

typical reaction of about 2 hours (using standard Taq polymerase)

visible via gel electrophoresis

analysed further – e.g. by restriction digestion, sequencing or cloning

Invented by Kary Mullis (1983) but basic principle of replicating a piece of DNA using two primers had
already been described (Gobind Khorana, 1971)
problem: limited by primer synthesis and polymerase purification issues

30–35 cycles
* Denaturation (95°C), 30 sec - DNA denaturation
* Annealing (55–60°C), 30 sec - primer annealing
* Extension (72°C) - primer extension (product duplication)

Question 2

requirements of begginind and end

Answer 2

unique sequences at the beginning and end of the genomic
region to be copied

Question 3

gene analysis prior

Answer 3

• Southern Blotting (1975): mapping of genes between unrelated individuals (RFLPs, insertions & deletions)
• DNA sequencing (1978): genes cloned into plasmid or ƛ vectors
• Gene library construction and screening: many months and each individual genome analysed

Question 4

contents of PCR (5)

Answer 4

Template DNA

Reaction buffer (Tris, ammonium ions [and/or potassium ions], magnesium ions, bovine serum albumin, [detergents])

Nucleotides (dNTPs)

Primers

DNA polymerase (usually Taq)

Question 5

Cycle 1-3 and characteristics of products, accumulation of PCR products

Answer 5

Primers delimit the length of the
majority of most of the PCR products, but a small percentage of products are of a non-specified length
small number of DNA copies whose length is undefined amplified from products of the first two cycles so only after the third cycle are all the subsequent products the desired length - products of undetermined length should be rapidly outcompeted by “correct” PCR products afte 3

As the cycle number approaches about 35 product accumulation
decreases until it plateaus
– The reagents are depleted
– The products re‐anneal
– The polymerase becomes damaged
Unwanted products accumulate and deplete reagents further.

Question 6

perfect reaction at 30 cycles how many copies

Answer 6

1,073,741,764 target copies (~1x10^9)

Question 7

automation

Answer 7

water baths
thermal cyclers only in 1986 - Perkin Elmer
E.coli used and had to be added after every cycle (not thermostable)
37°C : caused non‐specific priming, resulting in unwanted products
Taq only 1988

Question 8

Optimising PCR

Answer 8

1- Confirmation of successful amplification (gel electrophoresis, size that you expected?, more than one band? any band the correct size?) - May need to optimize the reaction conditions

2- Target and reagents concentration:
- Annealing temperature of the primers: theoretical, confirmed practically, 2°C above and below are tested, below the optimum primers bind to unintended sites

• concentration of Mg2+: fidelity because polymerase recognizes DNA bound to Mg2+, too much - less specific, too little- less yield, vary in 0,5mM - compromise between yield and specificity
• Reducing template and polymerase increases competition for resources: enough to complete the reaction but scarce enough to limit unintended reactions - enzymes (follow recommendations) and DNA (around 10ng human, adjust with genome complexity)

(Less common)
- The extension time
- The denaturing and annealing times
- The extension temperature

3- Optimisation of PCR cycle stages

• Denaturation (usually 5-10min but not all kits use): denaturation of complex targets (GC) and secondary structure, activation of hot start enzymes (prevent extension of incorrectly bound primers during in the initial cycle)

-Extension: final extension usually >5min - partially copies completed, polymerases integrate A tail, stutters when analysing with detection of single base discrimination, vector A tail

Question 9

PCR fidelity

Answer 9

Polymerase fidelity: Taq has a higher error rate than more modern proof-reading enzymes
and mixes (error distribution will be random - if a large enough proportion of the reaction is being used, proof reading enzymes are not required)

importance of fidelity: when subsequent applications use only a sub-sample and interrogate the sequence (cloning or NGS), Pfu proofreading enzyme, Direct PCR product sequencing or restriction enzyme digestion will not
show individual errors as most PCR products will have the correct base for each site

Target site: usually 100 – 2000bp but can be 25Kb just need reaction conditions to be modified:
- to ensure sufficient time for copying completion
- consider enzyme replication rate when setting duration extension
step
- Higher quality starting genetic material

Question 10

RNA amplification and cloning PCR

Answer 10

PCR cannot directly amplify RNA as the polymerase is
DNA specific - must be converted to cDNA first by reverse transcriptase (some kits combine the reverse transcription and PCR)

Cloning of PCR:
Most non-proofreading Taq polymerases add an adenosine to the ends of the PCR products
TA cloning - no control of orientation

Question 11

primers

Answer 11

~20 bases with GC 45-55%
differ 1ºC from one another
not pair with each other (dimers) or themselves (hairpin)
3´should be G or C
must not anneal to repetive DNA elements
not have mismatches at 3´end and 5´end can have non target seq to adapt final products

primer design - eg Primer 3
we need to consider if other regions might also be amplified - consider the sequence of the entire genome unsing in silico PCR- the UCSC tool (searches the entire human genome looking for potential primer sites within 4,000 bases of one another, size, temperatures, amplified region)

avoid SNPs and other variants: only one allele might be amplified if: - SNPCheck3 (identifies SNPs and variants under the primers)
- a SNP occurs near the 3 ́ end of a primer
- a long length polymorphism (e.g. a LINE1 insertion) occurs within the
target region