PCR and its Role in Diagnostics Flashcards
What is the definition of 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 a chain reaction?
A Chain reaction is a series of events each one of which is dependant upon the preceding event to sustain itself.
Typically it is a series of reactions that lead to an exponential increase in the number of events occurring in a sequence.
What is PCR and how does it work?
A method to specifically amplify segments of DNA
The segment amplified is called the amplicon and is determined by the sequence at the end of the DNA that is being amplified
Specificity stems from complementarity of the primers
These molecules are short oligonucleotides that are complementary to the sequences at the end of the amplicons and are able to form duplex by hybridising to them.
DNA polymerase recognises these duplexes and forms an initiation complex around them
Thus the specificity of PCR is determined by the uniqueness of the sequences at the end of the amplicons and the complementarity of the primers to these sequences
Secondly specific only if annealing is undertaken at the melting temperature Tm of the primers, i.e. high stringency conditions
This prevents mis-matched based pairing
Recall the steps for PCR.
(1) denaturation, in which double-stranded DNA templates are heated to separate the strands (around 90C)
(2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA (around 56C)
(3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA (72C)
What does the PCR reaction require?
A template strand with a primer (usually 20-30 bases long) annealed to it
Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
Mg2+ ions- essential cofactors for all DNA polymerases
A roughly neutral pH
How/ when does the chain reaction end in PCR?
Every cycle results in a doubling of the amount of product thus is an exponential accumulation of product
The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction
The plateau is caused by the acidification of the reaction that happens when the strands are elongated.
What are some of the applications for PCR?
Diagnostics :
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
What is q-PCR and what can it be used for?
PCR can be quantitative; real-time PCR- qPCR
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 at the end of each cycle of PCR by placing a threshold
Are used for quantifying the amount of target DNA molecule in the sample
How can q-PCR be used to detect SNPs and for what purpose
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 (see earlier lecture)
Common Applications:
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 are the two approaches to SNP detection?
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 can microsatellites be used in forensics?
Forensic identification uses repetitive sequences (STRs) within the human genome
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- i.e. the number of repeats varies between individuals
Provide a pattern of uniquely sized products accorded by each individuals genome but are inherited, one from your mother one from your father
Provides something akin to a molecular bar code or “DNA fingerprint”
UK DNA database currently consists of 10 STRs
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
