Polymerase Chain Reaction Flashcards
When was PCR devised and by who?
In mid 1980s by Kary Mullis - while on LSD
What is the principle of PCR?
This method is an in-vitro enzymatic method for amplifying exponentially a specific pre-selected fragment of DNA
What certain features does PCR exploit of DNA replication?
-DNA polymerase uses a single stranded DNA as a template for the synthesis of a complimentary new strand
-DNA polymerase requires a small section of double-stranded DNA to initiate or “primer” synthesis
What reagents must be placed in a tube to achieve in vitro DNA replication or PCR?
-Need a sample of DNA that we think will have the sample we want to amplify
-Enzyme - DNA polymerase
-DNA primers to direct polymerase to where it needs to go in DNA (template)
-Source of nucleotides - building blocks
-Cofactors to support enzymes - e.g magnesium
-Buffer
What is DNA amplification by PCR achieved by?
-A source of DNA (template)
-DNA polymerase enzyme
-2 synthetic oligonucleotide primers
-4 standard deoxyribonucleoside triphosphates (dATP, dGTP, dTTP, dCTP)
What are the 3 steps of PCR amplificationß
- DEnaturation
- Annealing
- Extension
What happens in each step of PCR?
- Denaturation - A DNA template is first denatured by heat - involves strand separation of the double stranded DNA (could use helicase enzyme)
- Annealing- Specific oligonucleotides are annealed to 2 separate sites on opposite template strands - primer- orientation important as will go opposite way if incorrect - dictate what part of sequence will be amplified
- Extension - extension of primers by polymerase-mediated nucleotide additions to produce 2 copies of the original DNA sequence
Why is this cyclic process repeated?
Repeated numerous times to produce a double-stranded copy of the original DNA fragment defined by the oligonucleotide (primer) binding sites
-Dont get exact target sequence at first few rounds cause enzyme doesnt know where to stop
-In 3rd cycle - target sequence synthesised right
Draw out 3 cycles of PCR
What are the 2 types of DNA amplified?
-One type produced in the first PCR cycles where the original template is copies to generate a strand beinning at the 5’ end of the oligosaccharide primer and ending only when polymerase ceases to function - few of these
-Second type of product is defined by both the 5’ and 3’ ends as the synthesis is terminated when the polymerase reaches the defined end of the template
What is the typical cycling protocol?
-95 deggrees for 7 mins - pre-denaturation
Denaturation - 92 degrees for 30s
Annealing 55 degrees for 1 min
Extension - 72 degrees for 2 mins
(32 cycles)
72 degrees for 5 mins (completion)
4 degrees holding
What is the starting material for a PCR?
DNA that contains the sequence to be amplified - small amount is needed
Often total genomic DNA extracted from cells but doesnt require highly purified DNA
Is it necessary to isolate the sequence to be amplified?
-No because it is defined by the primers used in the rxn
Is DNA stable or unstable
DNA is very stable and can last very long if hasnt come into contact with nucleases e.g DNA isolated from mummies and amplified by PCR
5 ingredients of a standard PCR
-Thermostable DNA polymerase
-Mg2+ ion conc
-Buffer
-PCR enhancers
-dNTPs
-Primers
What was originally used as the DNA polymerase and what is used now instead in PCR?
-Originally Escherichia coli DNA polymerase but was heat-sensitive and destroyed at temps used to separate ds DNA so had to add fresh enzyme manually for each cycle
-Heat stable Taq DNA polymerase isolated from Thermus aquaticus (bacterium lives in 75 degrees water, polymerase optimum of 72 and is stable still at 94 degrees)
-Automation of processing can be used since Taq polymerase only has to be added at the start
How is specificity and sensitivity of PCR improved by Taq polymerase?
-At lower temps required for e-coli polymerase, primers could anneal at sites where sequences differ slightly from target sequence
What does in vivo DNA polymerase have that in vitro Taq polymerase doesnt have?
Taq DNA polymerase doesnt have proofreading capability - typically this enzyme can incorporate one incorrect nucleotide for about every 2 x 10’4 nucleotides added
-Can acquire proofreading thermostable polymerases but so expensive
Why is a Mg2+ ion concentration needed in PCR?
Taq DNA polymerase is sensitive to divalent cation concentrations
A conc of 2mM MgCl2 is optimal
-Higher Mg2+ ion conc are inhibitory
Why may the precise Mg2+ conc need to be inhibited?
As both the oligonucleotide primers and the dNTP’s substrates for Taq will bind Mg2+
What is used in the buffer composition?
Modest concentrations of KCl can stimulate Taq DNA polymerase activity by up to 50-60% at 50 mM
-Higher conc will inhibit and activity lost at greater than 75mM
Examples of PCR enhancers?
Some PCR assays include enhancers to improve PCR amplification of target sequences such as urea, formamide and DMSO
-helps promote denaturation of complex region in target material - may need to add enhancer if PCR not working
How are dNTPs added?
In excess
Mix of all 4 added in same conc
How do primers affect the PCR?
-The sequences and combinations of primers determines the ultimate outcome of a PCR assay
-Useful primer lengths are between 14-20 bses in length w a 50% GC content
-Aim is to maximise both efficiency and specificity of amplication rxn
