L7 PCR Flashcards
What is PCR
Replication of DNA efficiently, rapidly. From a tiny amount of sample into a load of samples.
Uses principles of DNA replication
Requirements of PCR
Based on DNA replication, uses 2 specific primers that bind both DNA strands in opposing directions
Outline PCR reaction
Double stranded DNA > Denatured to obtain single strand DNA > Annealing step - Primers are designed to bind specific locations to target sequences. 40-65 degrees > Extension - DNA polymerase performs DNA polymerisation > A copy of DNA strand is made. > DNA polymerase will continue to add nucleotides hence will have a longer copy than original. > A third sequence (replication) will allow to copy to original target sequence.> The more the DNA is replicated, the more reliable it is due to closer copying.
How to verify PCR?
Gel electrophoresis. Using a size marker and comparing.
Conditions/requirements for PCR?
Up to 35 cycles. Apparatus must prevent condensation, hence temperature must be constant throughout entire equipment.
(PCR) Template
Template - any DNA, the amount is critical, too much may result in non specific amplification. How much DNA? - depends on the complexity of the substance. i.e genomic DNA - use high concentration. Plasmid DNA - lower concentration. DNA needs to be clean!!
(PCR) DNA polymerase
- Thermostable polymerase - maintain activity at high temperatures.
*Proofreading activity - 3’ -> 5’ proofreading activity
‘Taq polymerase’ does not have 3->5 proofreading activity. Pfu polymerase has proofreading.
Non proof reading polymerase make non-blunt ends, proof reading is oposite (blunt ends)
(PCR) Primers
- Single strand oligonucleotides, that compliments the target sequence. 15-30 bp, 40-60% G+C content.
- Bad primer - has mutiple groups with the same sequence hence they will form a “Hairpin”. Also primers that easily bind to each other.
- Good primer - must have a perfect match at the 3’ end of the primer relative to the DNA strand.
Anealing step
Needs to have a very specific temperature to allow primer to join. The optimal temp is 2-5 degrees below the melting temperature (Tm).
TM = [(A+T) x 2 + (G+C) x 4]
Primer modification
Primers can contain a restriction site, at the 5’
Cloning PCR products
non proof reading will have A nucleotide overhand. This can be used for cloning purposes. This can be ligated to a vector with a T overhang (complimentary).
PCR reaction conditions
Standard components-
20mM Tris/HCL, PH 8.3
10mM Kcl
2 mM MgCl2
K and Mg bind to the phosphate backbone (-ve charge). This helps with annealing the DNA
Applications of PCR
Probes for hybridisation
Assay for the presence of infectus agents
Direct cloning from genomic DNA or cDNA
Quantification of rare or low abundance of DNA and RNA
In vitro mutagenesis
DNA cycle sequencing
Genetic fingerprinting for forensic samples
Analysis of allelic sequence variations
Prenatal diagnosis of genetic diseases.
Diagnosis of Genetic disease via PCR
Need to know genetic elements, what genes associated etc.
PCR allows rapid genotyping of genetic markers
Use to detect deletion/insertion or point mutations
Templates: Blood, body fluids,hair,sement,sweat etc.
Eg. Detection of restriction site polymorphism.
1) Use Primer that target sequence of interest
2) Amplify by PCR
3) Cut strand w/ restriction enzyme, normal gene will be cut by site of interest, the mutated gene will not be cut.
4) Compare via gel electrophoresis.
E.g 2- Microsatellites - Huntingtons disease
Normal 10-35 CAG, Mutant 26-121 CAG repeats
1) Use primer to amplify either side of the repeats
2) Gel electrophoresis
3) Mutant has higher band distance.
E.g 3 - Sickle cell anemia
Mutation in haemoglobin gene, exon 1
1) 2 primers are used primer 1 or 2, having A, T ends respectively, theyre used simultaneously.
2) Normally, only primer 1 will react, primer 2 will not work.
E.g 4 - Tuberculosis
Genome containing several variable number tandem repeats units (MIRUs)
PCR amplification will categorise sizes of each repetitive units.
