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Flashcards in 3. PCR and Its Role in Diagnostics Deck (23):

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 a chain reaction?

A Chain reaction is a series of events each one of which is dependant upon the preceding event to sustain itself.
- leads to an exponential increase in number of events.


How is specificity sustained?

Only if annealing is undertaken at the melting temperature Tm of the primers, ie high stringency conditions. This prevents mis-matched based pairing.


How is the segment amplified determined?

The segment amplified is determined by the sequence at the ends and exponential amplification requires two primers. If we want to amplify a segment bounded by known sequence we can do this by using primers complementary to these ends.


How does DNA polymerase work?

• Complementary copy of the opposing template strand is synthesised.
• 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 (template annealed with short strand of ssDNA) from the 3’ end of the non-template strand.
- (Partially dsDNA is formed after annealing ss primer to a ss denatured template - stabilised by H bonds b/w bases).


How is annealing favoured over renaturation?

The annealing & renaturation are a competitive processes
- Template: low copy number
- Primer: very high copy number


Rules of DNA polymerase

• It synthesises a new nucleic acid strand by copying a DNA molecule.
• It cannot copy RNA nor make RNA
• RNA must first be copied to cDNA by reverse transcription before it can be amplified by PCR


PCR transitions between which 3 states?

• Denatured (template becomes single stranded)
• Annealed (formation of initiating template)
• Native state at the optimal extension temperature and pH for enzyme activity (for elongation)
* The transition depends upon hybridisation or primers and formation of a partial duplex.


Requirement of DNA polymerase

1) A template strand with a primer (usually 20-30 bases long) annealed to it
2) Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
3) Mg2+ ions
4) Enzyme needs to be thermostable - retain activity with repeated heat applied. ~ polymerase from a thermophilic bacterium often used (Taq polymerase).


Describe events of typical PCR cycle

1) Template, Primers, Enzyme & Reactants are mixed together
2) The template is denatured with high temperature.
3) Primers anneal with DNA at Tm of the primers.
4) The partially dsDNA extends from the 3' end of the primer
5) The reaction is reheated and cooled again. - Cycling typically 30-40 times.


Describe the kinetics of the reaction

• The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction.
• Sigmoidal shape of the curve because as the reaction progresses, the conditions are becoming more acidic. – consequences of the addition of the dNTPs to the elongating strand, and also producing pyrophosphate in the elongating process


Applications of PCR

1) Diagnostics
2) Diagnostics
3) SNP detection
4) Forensics and law enforcement ~ Amplification of genetic markers
5) Amplification prior to NGS and cloning or sequencing
6) Manipulating and modifying DNA
7) Important in recombinant DNA technology

3) Amplification of genetic markers ~in forensics and law enforcement: - parentage/kinship, identification, matching sources at crime scene, authentification of biology material (cell lines, food purity).
4) Amplification of material before NGS - e.g. Simultaneously sequencing large number multiple PCR products of candidate Cancer genes.
5) Amplification prior to isolating individual segments of DNA before cloning of sequencing
6) Manipulating and modifying DNA - e.g. introducing mutations or restriction sites.
7) Important in recombinant DNA technology - for vaccines and pharmaceuticals (interferons, clotting factor, tissue plasminogen activator etc).


How is PCR used in diagnostics?

Used for identification, confirmation and quantification (qPCR used) of specific DNA sequence.
• Presence absence calling TB - detection in sputum, determining treatment response/drug efficacy
• Differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes
• How much: determine when treatment might be commenced, “HIV viral load”


What is qPCR and why is it used? and how?

Real-time PCR or quantitative PCR
As there is the same end point of PCR, as amplification becomes rate limited - so it doesn't reflect the amount of template in the mixture.
To detect amount of target DNA used, real-time PCR (or quantitative PCR) is used (modification of PCR) ~ this is includes the use of fluorescent detection of the amplification.
- Serial dilution of the template produces curves shifted to the right (fluroescence detected at higher cycles).
- The crossing point of the amplification is determined and is proportional to the template conc. at the start.


How is PCR used for SNP detection?

SNPs are used as genetic markers for association studies.
* Two methodologies, (HRM and probe based version of qPCR), which are adaptations of quantitative real-time PCR. ~ They both depend on differences Tm conferred upon short sequences of DNA by their nucleotide composition ~ of a duplex containing a single nucleotide mismatch


• PROBE BASED VERSION OF qPCR (aka Allelic discrimination)


Examples of 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).


Applications of PCR in forensics and law enforcement in the amplification of genetic markers?

Amplification of genetic markers:
• Parentage or kinship: immigration and inheritance
• Identification: military casualties, missing persons or environmental disasters
• Matching two sources: crime scene
• Authentication of biological material: cell lines, purity of foods


What is a STR and why is this important in forensics? and how does this use PCR?

• STRs = Short tandem repeats ~ 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
• Forensic identification uses repetitive sequences (STRs)
• They are Highly polymorphic- ie the number of repeats varies between individuals, but they are still inherited so similar within a family.
• Provide a pattern of uniquely sized products accorded by each individuals genome providing a molecular bar code or “DNA fingerprint”
• UK DNA database currently consists of 10 STRs and each STR will differ in size
* The technique uses PCR with labelled primers that flank the STRs. The products are separated on a gel by size and the label is read to identify the size of the STR and which one of the 10 it represents


What are examples of process that PCR amplification material happens 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


How is PCR used to manipulate and modify DNA?

• Introducing mutations into a sequence of DNA
• Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors


Give examples of how PCR is used in recombinant DNA technology.

Developing recombinant vaccines, pharmaceuticals (interferons, clotting factors, Tissue plasminogen Activator etc)


How is HRM carried out?

• HIGH RESOLUTION MELTING (HRM) - Tm of the amplified product is used to determine which sequence is present. - Where differences in the amplified product is used to determine the presence of a SNP
• After PCR we produce a melt curve – heat up the reaction and slowly cool it and measure the annealing of the template
- The Tm is effected by a partcular sequence in the amplicon, forming different curves which describe particular variants within different amplicons.
- Comparing the curve with a know SNP in an amplicon will help to determine which SNP is present.


How is a probe based version of qPCR carried out?

• PROBE BASED VERSION OF qPCR (aka Allelic discrimination) where specific binding of the probe to the amplified region containing the SNP is detected. - Where differences in the Tm of a short probe spanning the SNP is used during qPCR
- Based on the depletion of the probe.