3- PCR and its Role in Diagnostics Flashcards
what is PCR?
an enzyme-based method to specifically amplify DNA segments called amplicons using a thermal DNA polymerase in a cyclical process - exponential doubling of molecules per stage of the reaction
reagents required for PCR and why
regents required: buffer, Mg2+ ions, dNTPs, DNA polymerase and template strands with annealed opposing primers
buffer = H+ ions are produced that can poison the reaction
Mg2+ ions = co-factor for DNA polymerase
thermostable (DNA dependent) DNA polymerase - often use Taq polymerase = must be able to undergo multiple rounds of heating and cooling
dNTPs = ACGT to form elongating strand
template strands with annealed opposing primers = one strand has a free 3’ OH and the other a 5’ overhang. can extend the strand from the 3’ OH
what are the 4 stages of PCR?
denaturation
annealing
elongation/extension
exponential amplification
briefly describe the 4 stages of PCR
denaturation - heating the DNA molecule at 95 degrees to break the H bonds, destabilise the duplex into single stranded molecules. Taq polymerase used as it’s a thermostable enzyme.
annealing - template-primer hybridising by cooling to predicted Tm of duplex. allows DNA poly. to recognise the partially ds structure and form an initiation complex.
elongation/extension - starting from the free 3’ OH end, adding nucleotides/dNTPs according to complementary base pairing at optimal pH and 72 degrees
exponential amplification - involves two primers and DNA poly. forming initiation complexes with each duplex to synthesise new strands
how is the specificity of amplification ensured?
uniqueness of the amplicon end sequence
specific complementarity of primers to end sequence
specific Tm of the specific template-primer duplex allowing annealing = uses high stringency conditions to avoid mismatched base pairing
describe in detail the process of annealing in PCR
amplicon is determined by its sequence of end bases - is the DNA sequence we want to copy
primers are made specifically to be complementary to the amplicon end sequence - have a free 3’ OH and 5’ overhang
surrounding temperature cooled to template-primer predicted Tm = allows annealing
high molar excess of primers means it outcompetes template renaturation - kinetics/equilibrium favours template-primer annealing
DNA dependent DNA polymerase recognises the partially ds structure and forms an initiation complex
what can affect/slow the PCR reaction?
acidification from production of H+ ions as nucleotides are added
depletion of dNTPs
what is quantitative (real-time) PCR?
qPCR allows us to obtain quantitative information about the amount of target DNA in a sample
how does qPCR work?
it works by measuring the fluorescence of the target DNA binding to the fluorescence probes per amplification cycle. the number of cycles it takes for fluorescence to reach a specific cycle threshold is used to estimate the initial amount of target DNA in the cycle via a standard curve. lower Ct = higher initial DNA amount
what modifications differentiate qPCR from PCR?
traditional PCR gives a qualitative result - indicates the presence or absence of a specific DNA sequence, but not the AMOUNT of that DNA. modifications include the fluorescent probes and real-time monitoring.
what is high resolution melting (HRM)? how does it work?
HRM is a post-PCR technique use to analyse the melting temperature of the DNA sample
DNA sample is amplified by PCR using fluorescent probes. after amplification, the temperature is gradually increased until it gets to a point where DNA starts to denature. as the temperature increases, DNA denatures and fluorescence decreases - data is plotted.
how are HRM and qPCR involved in detecting SNPs as real-time PCR techniques?
HRM uses the differential Tm of the amplified SNP to determine which sequence variant is present - compares to database.
qPCR uses a probe-based version involving allele-specific primers and fluorescently labelled probes which specifically bind to the amplified region within the SNP
what are SNPs and what are they used for?
SNPs are single base pair variations in a DNA sequence generated by mismatch repair during replication - used in genetic association studies
what is an STR?
a short tandem repeat specific to an individual in size and repeating bases. they’re scattered in specific locations throughout the genome and similar in close relatives.
how is PCR modified for the forensic use of STRs/microsatellite genetic markers? what is it used for - examples?
how it works:
1. a sample is collected, containing STRs specific to an individual
- multiplex PCR amplification with differently fluorescent-labelled priers that are specific to the flanking regions of one of the 10 STR loci
- capillary gel electrophoresis separates the DNA fragments of the 10 STR loci by size. a fluorescence sensor detects the intensity of fluorescence and plots an electropherogram .
- the peaks generated are distinct and specific to a certain STR locus. the more STRs investigated, the more unique and detailed the genetic profile produced - easier to identify the individual from the sample.
used in:
paternity tests
identifying missing individuals
