Essential Biomedical Research Skills - Practicals (Strand A) Flashcards
What is PCR?
-Polymerase Chain Reaction
-Amplification of DNA (exact copies)
-Based on DNA polymerase being able to synthesise a new strand of DNA complementary to the original ‘template’ strand.
-Starts with a single stranded piece of DNA.
-Uses Taq polymerase for repeated cycles.
-With each cycle there is an exponential increase in strands
-Essentially copies aspect of replication.
What is the prokaryotic model of PCR?
- DNA helicase separates dsDNA strands, binds to structure and energetically breaks the H bonds between them. Phosphodiester bonds do not break.
- Primases attach RNA primers to the open strands.
- DNA polymerase binds to double stranded structure and slides along leading strand of DNA, producing complimentary strand, from 5’ to 3’.
- Lagging strand, produce smaller fragments of DNA (Okazaki fragments) =pointless so ligase joins (ligates) these together.
What does alpha polymerase do?
Starts initial process on leading strand.
What does epsilon polymerase do?
Produces lagging strand
What does delta polymerase do?
Takes over to produce DNA product.
What is needed from replication process for PCR?
-Leading strand
-Template DNA
-Polymerases
Why use PCR?
- Sensitive – amplify as little as one molecule of DNA.
- Specific – can amplify a unique target sequence, stringency depends on temperature and Mg2+.
- Cheap
- Rapid – results available in a few hours.
- Robust – DNA is very stable and can be amplified from old and degraded samples.
What goes into PCR tube?
-Template – double stranded DNA, the sequence to be amplified
-2 primers – to prime synthesis in both directions (small ssDNA molecules, 6-30 bases, chemically synthesised)
-Polymerase – copies the template, extending from 3’ end of primer.
-dNTPS – deoxyribonucleotide triphosphates, dATP, dCTP, dGTP, dTTP
-Magnesium – essential for polymerase.
-Buffer – maintains optimal pH
-Water - makes up final conc
Nucleotide characteristics
-Building blocks
-Phosphate (strength), sugar (orientation), base (specificity)
-Thymine, Cytosine, Adenine, Guanine
Taq Polymerase characteristics
-All living cells contain polymerase.
-Copy DNA accurately
-Enzyme has 3 regions:
1. Synthesis
2. Proof reading
3. Primer removal
-Taq polymerase can withstand high temperatures.
Primers characteristics
-2 primers – one for each strand
-Single stranded DNA (oligonucleotide)
-Length of 18-24 bases:
* Too long – hybridise too slowly.
* Too short – won’t be specific, may bind elsewhere.
-40-60% G/C content
-Start and end with 1-2 G/C pairs.
-Melting temp of 50-60C.
-Primer pairs should have temp within 5C of each other.
-3’ end must be complementary to the template DNA
-Primer pairs should not have complementary regions.
Magnesium characteristics
-MgCl2
-A co-factor
-A non-protein component of the reaction that’s needed to enable the activity of the catalysis. Maybe metallic or compound based.
-Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application.
Buffer characteristics
-10x buffer
-Optimal pH is 8-9.5
-Tris HCl
-Potassium ions (KCl) – promotes annealing.
*May be replaced by ammonium sulphate, which destabilises base pairing bonds.
Describe PCR reaction
-Denaturation – break bonds by heating to 90C
-Annealing – primer binds to specific sites, temp reduced to 55C.
-Elongation – heat to 72C, optimal temp for Taq polymerase to operate.
1st cycle – synthesis of a strand of DNA in a test tube
2nd cycle – synthesis of 2 strands in a test tube
The rest – simultaneous synthesis of both strands
-30 cycles = >1 billion copies
Function of sliding clamp
Holds the polymerase on the ssDNA.
