PCR And Its Role In Diagnostics Flashcards
What is meant by the term “Polymerase Chain Reaction”?
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 series of events each of which is dependent upon the preceding event to sustain itself. Leads to an exponential increase in number of events.
Why is PCR so specific?
- Is only specific if annealing is undertaken at the melting temperature Tm of the primers
- This prevents mis-matched based pairing
What is the segment amplified determined by?
Determined by the sequence at the end
How would you amplify the segment bounded by the known sequence?
By choosing primers complementary to these ends. Exponential amplification requires two primers each complementary
What is the DNA dependent DNA polymerase enzyme used for?
- This 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
How is a partially double stranded structure formed in PCR?
By annealing a short single stranded DNA molecule (a primer)
How is this achieved?
- The double stranded template first has to be denatured and converted into a single stranded molecule
- Performed only after the template is denatured by heat
- The newly formed strand is sometimes referred to as the nascent strand
What is annealing of the primer to the template?
Annealing is an alternative way of describing hybridisation
How is annealing of the primer achieved?
Achieved using the predicted melting temperature of the primer template duplex under high stringency conditions
What does annealing result from?
Annealing results from the formation of base pairing, stabilised by hydrogen bonding
What type of process is annealing of the primers vs renaturation?
- A competitive process
- 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
What are some basic rules about PCR?
- Enzyme used is a DNA dependent DNA polymerase
- Synthesises a new nucleic acid strand by copying a DNA molecule
- It can’t copy nor make RNA
- RNA must be first converted to DNA by reverse transcription before it can be amplified by PCR
What does the PCR reaction require?
- A template with opposing primers
- Deoxy nucleotide triphosphates
- Mg2+ ions
- A roughly neutral pH
What is the PCR reaction based on?
The reaction is based on a transition between 3 states reliant upon hybridisation of primers and formation of a partial duplex
- Denatured (template becomes single stranded)
- Annealed (formation of initiating template)
- Native state at the optimal extension temperature and pH for enzyme activity
What must be required for the PCR reaction to work?
- The reaction must go through multiple rounds of extreme heating and cooling
- So the polymerases MUST be thermostable (able to retain activity)
What are the results expected from a PCR?
Every cycle results in a doubling of the amount of product therefore is an exponential accumulation of product
- The reaction has characteristic kinetics determined by depletion of reaction and the acidification of the reaction
What are diagnostic tools used for?
Used for identification, Confirmation and Quantification of specific DNA sequence
Give some examples where diagnostic tools are used?
- Presence absence calling TB - detection in sputum, determining treatment choice
- Differentiating between closely related organisms “Swine flu vs human influenza” both H1N1
Why can’t the PCR reaction inform the template copy number?
The end of the PCR reaction does not have a quantitative output and can’t be used to inform template copy number
What is the cause for this case?
- Same end point as amplification becomes rate limited
- This is independent of the starting concentration of template
How is this problem solved?
We therefore use modifications of this technique to provide measurable output during the exponential phase of the amplification in real time
What is quantitative PCR methods also known as?
There are a number of different quantitative methods which are collectively referred to as real time PCR or quantitative PCR
What do these Real time PCR techniques utilise?
- These techniques utilise fluorescent detection of the amplification
- Are used for quantifying the amount of a target DNA molecule in the sample
What does SNP stand for?
Single Nucleotide polymorphism
What are the methods that enable us to detect single nucleotide genetic variants?
Adaptations of quantitative real time PCR
What does these methods depend upon?
These methods depend upon the difference in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition
What are the common applications for these methods?
- Antibiotic resistance testing: TB and many other organisms
- Identification of genetic markers: Drug sensitivity/catabolism, markers of disease (cancer) or treatment response (HCV)
How many approaches are there for SNP detection?
Two
What are the two approaches for SNP detection?
- High resolution melting (HRM)
- Probe based version of qPCR
What is the high resolution melting (HRM) approach?
Tm of the amplified product is used to determine which sequence variant is present
What is the probe based version of qPCR approach?
Where specific binding of the probe to the amplified region containing the SNP is detected
How are genetic markers amplified?
- 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
- Authentication of biological material: cell lines, purity of foods
Describe Forensic use of micro satellite genetic markers
- Forensic identification uses repetitive sequences
- STRs are 2-5 or more bases in length repeated many times at specific locations in the genome
- They’re highly polymorphic: Yhe number of repeats varies between individuals
- Provides a pattern of uniquely sized products accorded by each individuals genome
What is forensics used for?
- Provides something akin to a molecular bar code or “DNA fingerprint
- UK DNA database currently contains 10 STRs
- Each STR differs in size, giving 20 numbers and a gender indicator
- Together they give a matching probability with an error of around 1 in 1 billion
How does the Polymerase Chain Reaction (STRs) look like?
VD
- 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 the other applications of PCR?
- Next generation sequencing: Sequencing large number multiple PCR products of candidate cancer genes
- Isolating individual segments of DNA prior to cloning or sequencing
What are the other applications of PCR? (Part 2)
Manipulating and modifying DNA
- Introducing mutations into sequences of DNA
- Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors
Why is PCR useful in todays society?
PCR is one of the most commonly used and important tools used in Recombinant DNA technology
