Outcome 3 Flashcards
What is PCR and what does it enable
Polymerase chain reaction - it enables the amplification of DNA fragments through repeated rounds of DNA synthesis
What are the requirements for PCR
Target DNA
Heat stable polymerase
Primers
dNTPs
Name the 3 stages of PCR
Describe the denaturation stage of PCR
The mixture of target DNA, heat stable DNA polymerase, dNTPs and primers are heated to 95C breaking the hydrogen bonds between bases of the dsDNA and seperating the dsDNA into ssDNA
Describe the annealing stage of PCR
THe mixture is cooled to 45-60C and primers which are present in excess bind via complementary base pairing to their recognition sites on the ssDNA. The recognition sites must be located at the 3’ ends that flank the target DNA sequence.
Describe the extension stage of PCR
The mixture is heated to 72C, at this temperature the taq polymerase uses the primer as its starting point for DNA synthesis. It catalyses the formation of phosphodiester bonds between the primer 3OH and the 5’ phosphate group of a dNTP that is complementary to the corresponding nucleotide of the target DNA to which the primer is bound.
What is meant by exponential amplification
PCR typically consists of 25-30 rounds. Each round produces double the amount of target DNA (exponential).
How can we confirm that PCR has worked
By agarose gel electrophoresis as the amplified target DNA should be of a defined fragment size.
What sets automated PCR apart from older PCR procedures
Thermal cyclers and taq polymerase.
What is a thermal cycler
A fully automated machine that can perform a round of PCR in about 5 minutes.
What is taq polymerase
A heat resistant polymerase with an optimum temp of 75-80C
What are primers
single stranded oligonucleotides that can bind to DNA through complementary base pairing and act as an initiation site for DNA polymerase to begin DNA synthesis
Why are 2 different primers used in PCR and what are they called
Forward primer - anneals to antisense strand
Reverse primer - anneals to sense strand
What are universal primers
Primers which are have a common nucleotide sequence complimentary to the base sequence of a particular set of DNA molecules. They can bind to a variety of DNA templates.
WHat are specific primers
Primers that bind specifically to a sequence of bases found at the 3’ end of a fragment of DNA of interest.
What factors is primers selection based on
Primer length melting temperature specificity complementary primer sequences base composition
Why is primer length a factor in primer selection
Specificity is partly based on primer length
If a primer is too short? non-specific binding possible
If a primer is too long? possible primers anneal to each other.
What is the typical range of primer length
15-24 bases
Discuss melting temperature with relation to primer selection
Melting temperature = temp at which half of the primer have annealed to the target DNA
BOth forward and reverse primers should have similar melting temperature that is above 60C
DIscuss complementary primer sequences
Primers should have low homology - low intra-primer complementarity to avoid hairpins
Primers should have low inter-primer complementarity to avoid dimers
WHat is ideal base composition for primers
40-60% GC
WHat factors decrease PCR efficiency
Contamination
Incorrectly selected primers
DNA polymerase
What steps can be taken to avoid PCR contamination
Set up PCR reaction and DNA analyses should take place in different rooms of lab
Dedicated pipettes are required for PCR, the pipettes should be bleached regularily
Gloves and lab coat should be changed after each stage of PCR
What is real time PCR
Based on conventional PCR, instead of analysing the final DNA yield with agarose gel electrophoresis. The DNA is quantified during the PCR reaction using fluouresence. The flourescence is acheived by either fluorescent dye which intercalates between bases or dye + labelled DNA probe which are oligonucleotide linked dyes. The flouresence intensity is measured after each cycle.
What are the advantages of PCR
As long as the nucleic acid sequence of the target genome is known, primers can be designed to amplify sections of it.
Sample preparation is automated
It provides results very quickly
It can be used to give a quantitative result (real time PCR)
What are the disadvantages of PCR
Cross contamination of samples from pipettes etc can give false positives unless strict decontamination procedures are employed.
Impurities in sample can interfere with PCR, sometimes purification of the clinical sample is needed