PCR and diagnosis Flashcards
what is PCR
a polymerase chain reaction which is enzyme based to amplify segments of DNA using a thermal DNA polymerase in a cyclical process
why is PCR specific if annealing is undertaken at the Tm of the primers?
prevents mismatched based pairing
what does the DNA dependent polymerase do?
recognises a specific structure consisting of a partially double stranded DNA.
forms a initiation complex with it
extends double strand from 3’ end of non template strand
how does PCR form a double strand structure from a single strand?
by annealing short single strand DNA
requires denaturing of double strand
what is the nascent strand
newly formed strand in PCR
what is annealing?
alternative way of describing hybridisation
explain annealing of the primer
occurs under high stringency
use predicted melting temperature of primer-template duplex
does the template have high or low copy number
low
does the primer have a high or low copy number
v high
annealing or renaturation?
annealing of primer occurs in preference to renaturation
driven by favourable kinetics
result of vast excess of primer present in reaction
describe the DNA dependent DNA polymerase used in PCR
- synthesises new nucleic acid strand by copying DNA molecule
- cannot copy rna or make RNA
- RNA must first be converted to DNA by reverse transcription before it can be amplified by PCR
rules for pcr
reaction is based on transition between states reliant on hybridisation of primers and formation of partial duplex
must go through multiple rounds of extreme heating and cooling
polymerase must be thermostable
what is thermostability
to retain activity, upon repeated heating to temps that would destroy most enzymes
example of thermostable bacterium
thermus aquaticus
describe 5 steps of PCR
- template, primers, enzymes and reactants assembled
- denature at 95 degrees
- anneal at the Tm of primers (55)
- extend from 3’ end of primer (72)
- 30 cycles of PCR = amplifies one single molecule to 1 billion copies
what are the applications of PCR
- diagnostics (identification, confirmation, quantification)
- differentiating between closely related organisms
- identifying individuals positive for covid and SARS
how can PCR be quantitative?
- used for diagnostics
- aka real-time PCR
- use fluorescent detection of amplification
- used to quantify amount of a target DNA molecule in sample
what is SNP?
single nucleotide polymorphism
describe SNP detection
- adaptations of quantitative real time PCR
- depend on difference in melting temp on short sequence of DNA
common applications of SNP detection
- antibiotic resistance testing
- identification of genetic markers
2 approaches of SNP detection
- high resolution melting = Tm of amplified product used to determine which sequence variant is present
- probe based version of PCR, specific binding of probe to amplified region containing SNP is detected.
uses of amplification of genetic markers in forensics and law enforcement?
- immigration and inheritance in parentage
- identification
- matching biological materials from two sources
- authentication of biological materials
PCR in forensics short random repeats
- identification uses repetitive sequences
- UK database consist of 10 STRs
- each STR differ in size, gender indicators
- error of 1 in 1 billion
other applications of PCR
- amplifying material prior to next generation sequencing
- manipulating and modifying DNA
- recombinant DNA technology
