W1 - Polymerase Chain Reaction (PCR) Flashcards
What is PCR?
Polymerase Chain Reaction is an enzyme
based method to specifically amplify segments
of DNA using a Thermal DNA polymerase in a
cyclical process.
What is Specificity?
It stems from the complementarity of the primers.
*Is specific only if annealing is undertaken at the melting
temperature Tm of the primers, ie high stringency conditions
*This prevents mis-matched based pairing
How does amplification work?
- Since the segment amplified is determined by the sequence at the ends
- If we want to amplify a segment bounded by known sequence we can do this by choosing primers complementary to these ends and exponential amplification requires two primers each complementary
What does DNA dependant DNA polymerase do?
The enzyme recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it.
The reaction extends a partially double stranded molecule from the 3’ end of the non-template strand.
What role does a primer play in DNA?
In PCR a partially double stranded structure is formed by annealing a short single stranded DNA molecule (a primer)
*In order to achieve this, the double stranded template has first to be denatured and thus made into single stranded molecule
*It is performed only after the template is denatured by heat
*The newly formed strand is sometimes referred to as the nascent strand
Primers hybridises with the sample DNA and defines the region that will be amplified
What is annealing and how is it involved in PCR?
*Annealing is an alternative way of describing hybridisation
*Annealing of the primer under high stringency conditions is achieved using the predicted melting temperature of the primer-template duplex.
*Annealing results from the formation of base-pairing, stabilised by hydrogen bonding
Why is denaturation favoured over renaturation?
Template: low copy number
Primer: very high copy number
* Annealing of the primer occurs in preference to renaturation and is driven by favourable kinetics as a result of the vast excess of the primer present in the reaction
Why is DNA polymerase required in PCR at all? What are the basic rules?
It synthesises a new nucleic acid strand by copying a DNA molecule.
* It cannot copy RNA nor make RNA
* RNA must first be coverted to DNA by reverse transcription before it can be amplified by PCR
What conditions are required for PCR to occur?
- A template with opposing primers (usually 20-30 bases long) annealed to the respective strands with a free 3’OH & 5 ‘ overhangs
- Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions
- A roughly neutral pH
What are the 3 basic stages of PCR?
The Reaction is based on transition between three
states reliant upon hybridisation of primers and
formation of a partial duplex
Some basic rules
*Denatured (template becomes single stranded)
*Annealed (formation of initiating template)
*Native state at the optimal extension temperature and pH for enzyme activity
Why is running strands through cycles necessary for PCR?
For PCR to work the reaction MUST go through multiple rounds of extreme heating and cooling
* so the polymerase MUST be thermostable
What is thermostability?
“able to retain activity” upon repeated
heating to temperatures that would “destroy” most enzymes.
Simply put, what happens in PCR?
1) There is a template, primers, enzyme and reactants
2) Denaturation occurs at 95 degree celsius
3) Annealed at the Tm of the primers eg. 55 degree celsius
4) Extend from 3’ of the primer 72 degree celsius
With 30 cycles = Over 1 billion copies
- exponential accumulation
- kinetics determined by acidification on the reaction.
What are some biological/ clinical uses of PCR?
Routine diagnostic tool used for identification, confirmation and quantification of specific DNA sequence
Examples:
* Presence absence calling TB - detection in sputum,
determining treatment choice
* differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes
* How much is present - determining when treatment might be commenced, “HIV viral load”
* Identifying individuals positive for SARS-CoV-2 and thus have CoVID-19
On a graph, what happens at the end of PCR?
The end of the PCR reaction does not have a quantitative output and cannot be used to inform template copy number.
Exponential graph plateaus.
We therefore use modifications of this technique to
provide measurable output during the exponential
phase of the amplification in “real-time”
What is the quantitative method of capturing PCR in real time?
*There are a number of different quantitative PCR detection methods used for diagnostics
* collectively they are referred to as real-time PCR or quantitative PCR
*These techniques utilise fluorescent detection of the
amplification
*Are used for quantifying the amount of a target DNA molecule in the sample
What is SNP?
Single nucleotide polymorphism SNP and single base alterations – single nucleotide genetic variants.
What is SNP detection?
*Several methodologies enable us to detect single nucleotide genetic variants, two are adaptations of quantitative real-time PCR
*These methods depend upon the differences in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition
What are common applications of SNP detection?
- Antibiotic resistance testing -TB and many other organisms
- Identification of genetic markers - drug sensitivity/catabolism (CYP2C9 and VKORC1 variants confer warfarin sensitivity),markers of disease (Cancer) or treatment response (HCV),
What 2 methods are used with SNP?
SNP detection
These two approaches are:
*High resolution melting (HRM) - Tm of the amplified product is used to determine which sequence variant is present
*Probe based version of qPCR (Sometimes referred to as Allelic discrimination) where specific binding of the probe to the amplified region containing the SNP is detected
How is SNP useful in forensics?
Amplification of genetic markers :
* Parentage or kinship: immigration and inheritance
* Identification: military casualties, missing persons or
environmental disasters
* Matching biological materials from two sources: placing an individual at a crime scene
* Authentification of biological material: cell lines, purity of foods
What are STRs and why are they useful?
Forensics Short tandem repeats (STRs or microsatillites)
* Forensic identification uses repetitive sequences (STRs)
* STRs are 2-5 or more bases in length repeated many times at specific locations in the genome
* Many different STRs are found scattered around the genome
* They are Highly polymorphic- ie the number of repeats varies between individuals
* Provide a pattern of uniquely sized products accorded by each individuals genome
Why are STRs important?
- UK DNA database currently consists of 10 STRs
- Provides something akin to a molecular bar code or “DNA fingerprint”
- and each STR will differ in size; giving 20 numbers and a gender indicator
- together they give a matching probability with an error of around 1 in 1 billion
- Multiple sets of labelled Primers are designed such that the products span different STRs
- The more STRs investigated the more unique the pattern of sizes produced providing a “DNA fingerprint” of STRs around the genome
What are other uses in PCR?
Amplifying material prior to:
* Next generation sequencing eg. Simultaneously sequencing large number multiple PCR products of candidate Cancer genes
* Isolating individual segments of DNA prior to cloning or sequencing
* Manipulating and modifying DNA
* Introducing mutations into a sequence of DNA
* Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors PCR is one of the most commonly used and important tools used in Recombinant DNA technology
* eg developing recombinant vaccines, pharmaceuticals
(interferons, clotting factors, Tissue plasminogen Activator etc)
