Lecture 8 - polymerase chain reaction Flashcards
PCR
Polymerase chain reaction
Highly specific and can be used to amplify, or copy specific DNA targets from a mixture of DNA molecules
Taq DNA polymerase
From thermophilic bacteria
Thermostable enzyme so can repeat PCR cycling without the need to add fresh DNA polymerase after each cycle
Basic requirements for PCR
DNA sequence of target region must be known
Template DNA for amplification
Primers- short oligonucleotides that will bind to a target sequence and prime DNA synthesis
Thermostable DNA polymerase which is not inactivated by heating to 95 degrees
dNTPs- deoxynucleoside triphosphate for each base
Thermal cycler- a machine that can be programmed to carry out heating and cooling of samples over a number of cycles
How many primers are required?
2 to flank the target sequence
Describe primer requirements
18-25 nucleotides in length
Similar Tm (50-65 degrees)
Similar GC content (which determines Tm)
GC clamp at the 3’ end to aid polymerisation initiation
Minimal secondary structure (avoids primers annealing to each other)
Minimal repeats
Minimal runs of a single base (no more than 4)
What is the main potential primer problem?
And what would the outcomes be?
Need to avoid self- complementarity or complementarity to another primer
Hairpins
Self-pairing
Inter-primer pairing
What happens if a primer annealing overhangs?
It could prime DNA synthesis to form primer dimers which are products formed by primers attaching to each other.
How do you prevent primer dimers?
Use less primer in the reaction Adjust the magnesium chloride concentration Use more template DNA Adjust annealing temperature Redesign primers
Describe primer dimer formation
In the annealing stage two primers, which have complementary parts, anneal with each other
Taq polymerase then elongates the strands to form complementary dsDNA. This is a primer dimer
A primer dimer can be recognised in an electrophoresis when you ding 2 bands of DNA when you only expect 1. The lower band is the primer Dimer.
What is seen with multiple cycles of PCR?
Exponential multiplication
Target DNA becomes isolated and duplicated
Increased risk and possibility of mutations
What is DNA, to be amplified, mixed with?
deoxyribonucleotides
thermostable polymerase
DNA primers
Step 1 of PCR
DENATURATION
90-95 degrees
The double stranded DNA breaks apart to single strands allowing access for Taq polymerase and primers
Step 2 of PCR
Annealing
55-65 degrees
Oligonucleotide primers bind to the regions next to the target gene which is being amplified. Primers are at higher comcentrations than the template target so they preferentially anneal to the template rather than the two strands re-annealing
Step 3 of PCR
Extension
68-72 degrees
optimum temperature for DNA polymerase. Taq polymerase will fall off when it reaches the end of the template
How do you confirm that the PCR has amplified the desired product and is the correct size?
Load onto an agarose gel
Hyper-ladder which contains DNA molecules of known size is loaded onto the gel
Larger molecules move more slowly
DNA is visualised by staining with ethidium bromide that fluoresces when exposed to UV light
What are the different variations of PCR?
Reverse transcriptase PCR Real time or quantitative PCR Assembly PCR or polymerase cycling assembly Allele specific PCR Methylation specific PCR
Reverse transcriptase PCR
Makes a complementary DNA copy of RNA by reverse transcription followed by PCR. Can be used to determine quantities of mRNA or genome copies of RNA viruses
Real time or quantitative PCR
A variation of RT-CR where the accumulation of PCR products is monitored through each cycle of the PCR
Provides accurate quantification of mRNA levels
(Polymerase cycling) Assemble PCR
Overlapping primers used to assemble into large chunks of DNA
Can be used to make synthetic genes or genomes
Allele specific PCR
Primers are designed that will amplify specific alleles of a gene
Methylation specific PCR
Distinguishes between methylated and non-methylated DNA
How can PCR be used as a tool in genetic fingerprinting
DNA profiling can be used to identify individuals
Small samples of DNA can be isolated from a crime scene to be compared with DNA from suspects or with a DNA database
PCR & molecular archaeology
Ancient DNA can be isolated from archaeological specimens. DNA tends to degrade gradually even when maintained in freezing conditions (permafrost)
Advantages of PCR
Sensitive, specific, rapid identification
Why is there no product generated until the 3rd PCR cycle?
After cycle 1 you have a copy of the amplified single stranded DNA with either the forward or reverse promer at each end and each are extended beyond the other primer position.
At cycle 2 you have extended sections but now the opposing primer can bind.
At cycle 3 you begin to make the blunt ended target that has each primer at the 5’ end of each strand of the dsDNA product.
