PCR Flashcards
What are the three steps of PCR?
Denaturation, annealing and extension
What happens in denaturation?
The double strand DNA templates are heated to separate the strands.
What happens in annealing?
Primers bind to flanking regions on the target DNA.
What happens in extension?
DNA polymerase extends from the 3’ end of each primer along the template strand.
How many cycles of PCR are usually done?
25-35
In what direction is the DNA amplified?
5’-3’
Why are Klenow fragments of DNA polymerase I from E coli no longer used in PCR?
As they are heat sensitive and so are destroyed at the high temperatures required for PCR
Which DNA polymerase is used now?
Taq DNA polymerase from Thermus aquaticus.
What are the drawbacks of Taq Polymerase?
It is unstable above 90 degrees and doesn’t proofread
What is the function of thermocyclers?
It automates temperature cycling and incubation times.
When were thermocyclers first introduced?
1985
From what sources can the template DNA be taken?
Any source i.e. genomic DNA, complementary DNA or plasmid DNA
What is the equation for copy number?
Avogadro’s constant x no. moles
What are the 7 components to a PCR reaction?
- PCR buffer
- dNTPs
- MgCl2
- Primers
- Taq DNA
- Sterile distilled water
In what order should the components be added to the PCR solution?
Start with the material that has the highest volume and work through to the lowest.
How long are primers?
Around 15-30 bases
What is the usual Tm range?
55-70 degrees and the two primers must be within 5 degrees of each other.
What should be avoided in PCR?
- Hairpin DNA
- Self dimers
- Repeats of more than four nucleotides
- GC-rich ends
What are the optimal conditions for PCR?
- 15-30 nucleotides long
- A Tm of 55-70
- 40-60% GC
- One G or C at the 3’ end
In what proportions are the four nucleotides added to the PCR solution?
In equimolar quantities
When may an unbalanced concentration of dNTPs be used?
In order to promote a higher degree of mis incorporation by non-proofreading DNA polymerase.
What is the recommended concentration of each dNTP?
0.2 mM
Why is a higher concentration of dNTP used in the presence of Mg2+?
As Mg2+ binds to the dNTPs and reduces their availability for incorporation.
With what are dNTPs sometimes replaced?
dUTP
What is the function of magnesium in PCR?
It has cofactor activity with DNA polymerase enabling incorporation of dNTPs during polymerisation.
What does the Mg2+ active site catalyse?
Phosphodiester bond formation between the 3’ OH primer and the phosphate groups of the dNTPs.
What is the function of buffers in PCR?
To provide a suitable chemical environment for the activity of DNA polymerase.
What pH is buffered in PCR?
pH 8-9.5
What is pH stabilised by in PCR?
Tris-HCl
What is reverse transcription?
When RNA is isolated from a sample and mRNA is converted back to cDNA
Why is endpoint PCR performed?
To quantitate RNA expression.
What can PCR be used to detect in genotyping?
Sequence variations in alleles of specific cells.
How is the primer altered for use in genotyping?
It is designed to flank regions of interest and assess genetic variations based on the presence or absence of amplicons and/or their length.
What is sequencing of PCR amplicons used to study?
Single nucleotide variations and single-nucleotide polymorphisms.
What can PCR be used to clone?
DNA fragments of interest.
What happens in direct PCR cloning?
Desired regions of the DNA source are amplified and inserted into specifically designed vectors. Or primers with extra nucleotides at 5’ end.
Why is it recommended to purify the DNA sequence?
To remove excess reagents and non-full length oligonucleotides.
What is PCR cloning used for?
To screen whether colonies carry a desired insert.
What is mutagenesis?
Cloning to introduce desired mutations.
What is site directed mutagenesis?
Primers that incorporate base substitutes, deletions or insertions.
What important considerations are needed in site directed mutagenesis?
Primer design, DNA polymerase choice and RNA parameters.
Why are high-fidelity PCR runs recommended when preparing sequencing templates?
To maintain DNA accuracy
What is Sanger sequencing?
When PCR amplifies fragment of purified DNA and subjects them to sequencing reactions.
How is the DNA altered in sequencing?
Primers are tagged at the 5’ end with binding sites for sequencing primers.
