PCR and its role in diagnostics Flashcards
what is PCR
polymerase chain reaction is an enzyme based method used to amplify specific segments of DNA using DNA dependent DNA polymerase ina. cyclical process
what is a chain reaction
its a series of events each one of which is dependant upon the preceding event to sustain itself - (series of reactions that lead to exponential increase in the number of events)
what is the segment that is being amplified called and how is it determined
amplicon - determined by sequence at ends of the section of dna that is being amplified
What determines the segment to be amplified by PCR?
2 primers (oligonucleotides) that are complimentary to the bases at the ends of the amplicon are able to form duplex by hybridising to them
what makes PCR so specific and under what conditions?
Specificity stems from complementarity of the primers.Is specific only if annealing is undertaken at the melting temperature Tm of the primers
ie high stringency conditions
- prevents mismatched base pairing
what conditions must PCR be in to ensure specificity
must be undertaken at a Tm near to the primers Tm, so under high stringency conditions
this avoids mismatches
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difference between probe and primer
primers are necessary for DNA replication while probes are necessary for detection of specific sequences in the sample. probes are labelled with fluorescence and detected in the annealing and extension phase
What is DNA Dependent DNA Polymerase?
a heat resistant and thermophilic enzyme that recognises a specific structure formed by partially double stranded DNA molecule with a 3’ end and will form an initiation complex with it
describe the whole procedure of PCR:
denaturation occurs first, at a high heat so the DNA template separates into single strands. short DNA primers anneal to complimentary matches on the single DNA strands under conditions of high stringency - near Tm (melting temp) of the primer, to avoid mismatch pairs and have high specificity.
and lastly we have native state at the optimal extension temperature and pH for enzyme activity
- extension by DNA dependant DNA polymerase which forms initiation complexes and extends the strand in the 3’ direction with a 5’ overhang by adding nucleotides
forming nascent strand
As annealing and renaturation are competitive processes, which is preferred over the other?
Template is at a low concentration at the start of the reaction
Primer is at very high concentration
So there is a huge molar excess of the primer - annealing of the primer occurs in preference to renaturation and is driven by favorable kinetics as a result of the vast excess of the primer present in the reaction
How does the primer-template duplex form
Formation of primer template duplex is forced to occur by providing a huge excess of primer
How much template strand is used?
The template is at the start of the reaction in a low concentration
What other reactions are competing with the formation of the primer-template duplex?
In competition between renaturation of double stranded template and the annealing of the primer to template
Outline how DNA Dependent DNA Polymerase works
It synthesises a new nucleic acid strand by copying a DNA molecule
It cannot copy RNA nor make RNA
How is RNA converted to DNA for PCR?
by reverse transcription - reverse transcriptase
What does DNA polymerase require to function?
- Template strand + primer annealed (20-30 bases long)
- Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- Mg2+ ions (cofactor)
- Roughly neutral pH
Why is it significant for the polymerase in PCR to be thermostable?
For PCR to work the reaction MUST go through multiple rounds of extreme heating and cooling
so polymerase MUST be thermostable
What is thermostability?
“able to retain activity” upon repeated heating to temperatures that would “destroy” most enzymes
Where do we obtain the thermostable polymerase used in PCR?
from a thermophilic bacterium such as Thermus aquaticus is often used (Taq polymerase)
Outline the cyclical process of PCR in detail
- Mix all reactants together
e. g. Template, Primers, Enzyme & Reactants - Denaturation: heat to thermally denature template
strand (95°C) - unwound due to heating, breaking H
bonds => separating strands - Cool mixture to temperature approximating primers Tm
(e.g. 50°C) - allows corresponding primers to bind to
complementary template strand - Heat to 72°C - optimal temperature for thermal
polymerase to work => an initiation complex forms as
polymerase recognises partial duplex, elongating 3’
prime end of primer to create a new strand
5.Repeat process multiple times (typically 30-40 times)
How much product is formed per cycle of PCR?
Every cycle results in a doubling of the amount of product, thus the exponential accumulation
Describe the kinetics of PCR reactions
The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction
Describe kinetics we would see on a linear scale when plotting the PCR reaction
would see a sigmoidal curve
as elongation occurs, H+ ions are produced due to addition of dNTPs to elongating strand (eventual plateau)
Also produce pyrophosphate
Why does the PCR kinetic graph Plateau?
No. of primers present depletes and [template] increases ∴ changing environment in which polymerase works as a consequence of acidification of the reaction causing a plateau
=> no longer producing product
What are the diagnostic applications of PCR?
identification, confirmation and quantification of specific DNA sequence
Outline examples where PCR is used in diagnostics
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”
Why is it difficult to ‘measure’ PCR
End of the PCR reaction doesn’t have a quantitative output
Can’t be used to inform template copy number or how much we started off with
Why is PCR end point not quantifiable?
Same end point regardless of starting concentration as amplification becomes rate limited
What does PCR graph represent?
no. of molecules produced during PCR (always unclear due to depletion of reactants) x no. of cycles
position of the curve reflects starting template copy number - can use this to measure increase in yield in real time
curve shifts more and more to the left when the starting copy number is higher
How do we quantify PCR if the end point isn’t measurable?
We therefore use modifications of this technique to provide measurable output during the exponential phase of the amplification in “real-time”
referred to as real time PCR or qPCR
What are the quantitative PCR methods available for diagnostics?
Collectively referred to as real-time PCR / Quantitative PCR
These techniques utilize fluorescent detection of the amplification used to quantify the amount of a target DNA molecule in the sample
allows us to monitor the early part of the reaction - when the product is detectable - reflects the starting point.
What is a SNP?
Single nucleotide polymorphism SNP – single nucleotide genetic variants
some are bad ie, sickle cell anaemia
How can we identify SNPs?
Several methodologies enable us to detect SNPs two are adaptations of quantitative real-time PCR
How do SNP detection methods allow us to identify and differentiate between SNPs?
These methods depend upon the differences in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition
rely upon effect on the melting temp (Tm) of a duplex containing a single dna mismatch
What are the common applications of PCR SNP Detection?
Antibiotic resistance testing -TB + 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 2 approaches to detecting SNPs?
High resolution melting (HRM): relies on effect that differences in amplified product with the variant i it has on the melting curve for a given sequence
Probe based version of qPCR (allelic discrimination):
where specific binding of the probe to the amplified region containing the SNP is detected
How is PCR used in forensics?
PCR is used in forensics and law enforcement in the amplification of genetic markers
Outline the uses of genetic markers and PCR in law and forensics
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, food purity
How does forensics allow us to identify?
Forensic identification uses repetitive sequences - short tandem repeats (STRs) or microsatellites
What are short tandem repeats (STRs)?
2-5 or more bases in length repeated many times at specific locations in the genome
Where are STRs found?
Many different STRs are found scattered around the genome
They are Highly polymorphic- ie the number of repeats varies between individuals
What is the significance of STRs?
act as a genetic fingerprint, differentiating person to person - every persons is different, but similar with siblings and parents as number of repeats differ!
How are STRs used to identify people?
Multiple sets of labelled Primers are designed such that the products span different STRs
More STRs investigated = more unique pattern of sizes produced providing a “DNA fingerprint” of STRs around genome
How common are STRs?
Each STR has a normally observed range in population
E.g.,
VWA is found on chromosome 12 consists of a TCTG or TCTA repeat, that is repeated between 11 and 24 times
What are the other applications of PCR and STRs?
Next generation sequencing
Manipulating and modifying DNA
What is the significance of PCR in recombinant DNA technology?
developing recombinant vaccines, pharmaceuticals
