4 - PCR Detection Methods

Types of PCR

• Allele specific PCR

• Conventional PCR

• Digital

• Multiplex

• Nested

• Real time

• Reverse transcriptase

Main principles of PCR

• New chain requires primer as polymerase can only extend existing double strand, oligonucleotides used, approx 20

• Extension only proceeds in 5’ to 3’ direction (polymerase only adds to 3’)

• Two strands are anti-parallel

Requirements for conventional PCR

• Target DNA (template)

• Buffer, magnesium (co-factor)

• Thermostable DNA polymerase

• Deoxynucleotide triphosphates (dATP, dTTP, dCTP, dGTP)

• Forward and reverse primers complementary to target sequence

Allele specific PCR

• Detect specific mutations e.g. cystic fibrosis CFTR gene

• DNA polymerase distinguishes between match and mismatch at 3’ end, primer designed with mismatch base corresponding to mutation

• Sickle cell caused by mutation in HBB gene which codes for B-globin (glutamine to valine)

• To detect, one common primer is used and other specific for one allele (A WT, or T mutant), two separate reactions set up

Nested PCR

• Variation of standard PCR that enhances specificity and yield of amplicons

• Two primers used, outer primers flank region of DNA containing amplicon of interest, nested primer corresponds to the precise region within the DNA to be amplified

• Outer primers used in first round, product serves as template in second round with nested primers

Multiplex PCR

• Allows for concurrent amplification of multiple targets with different primers

• Requires two or more probes that can be distinguished from each other

Real time PCR (q-PCR)

• Allows for analysis of PCR products in real time, allows for quantification in exponential phase, very sensitive

• LightCycler Pro

• Two types of flourescent technologies can be used - intercalating dyes or 5’ nuclease probes

Intercalating dyes

• Incorporate into minor grooves of dsDNA and only then do they fluoresce

• Emission will increase in each cycle during the extension phase, low or absent during denaturation

• Advantages - can be used with any pair of primers, cheaper and requires less knowledge than 5’ nuclease probes

• Disadvantages - specificity diminished due to risk of amplifying non specific products or primer dimers, melt curve analysis must be performed to distinguish between specific and non specific products

Melt curve analysis

• Temperature gradually increased from 60 to 95, fluorescence monitored continuously

• High emission at low temperatures when PCR products are double stranded

• Products of different lengths or GC content will have different melting temperatures creating distinct peaks

• Primer dimers have low melting temperature (peak at lower temp)

5’ Nuclease probes

• Double labelled oligonucleotide probes with two reporter and quencher fluorochromes which emit a signal upon cleavage

• Based on principle of fluorescence resonance energy transfer (FRET), energy transfer between two light sensitive molecules

• Non-extendable at 3’ end, designed to anneal to target internally of the primers

• Upon binding, probe is degraded by exonuclease activity of polymerase

• Increase in emission directly proportional to amount of product

Data acquisition

• Point in exponential phase when amplification of product reaches detection level

• The more input DNA the less cycles it will take to make a specific amount of amplicon

• Threshold is chosen based on the variability of the baseline, 10 times the SD of baseline

• Threshold cycle (ct) values are then calculated by determining the point at which fluorescence exceeds this threshold

Multiplex PCR

• Good if there is a limited amount of input material

• Up to 6 different colours

• Limited to to existing knowledge of microorganisms genome, no distinction between live and dead organisms, unexpected mutations not detected

• Used in combination with cultures

Reverse transcription q-PCR

• Used for quantification of mRNA expression

• RNA must be good quality, consistent conditions, absence of co-amplifying genomic DNA

• Normalisation - correction for experimental variations in RT and PCR amplification efficiency, reference gene used

RT q-PCR method

• RNA converted to complementary DNA (cDNA) by enzymatic RT reaction

• Creates complementary strand of DNA based on RNA sequence, DNA primer required, ssDNA then used as template to synthesise dsDNA (cDNA)

• Entire mRNA sample can be converted to cDNA by oligo(dT) primers that anneal to poly A tails of mRNA

• Random hexamers can also be used, primers 6 nucleotides in length, randomly bind to sample at any location

Data quantification using a standard curve

• Sample of a known concentration is used to make serial dilutions

• Ideal slope is -3.3 (up to 3.6) for 10 fold dilutions, measures efficiency

• CT values are plotted against quantity, unknown amount of input can be measured

Data quantification using the comparative threshold cycle method

• Calculate relative expression levels compared to a calibrator (control)

• Value of unknown target is normalised to a reference gene

• The efficiency of the PCR amplification for the target and reference gene must be approximately equal

Digital PCR

• Based on limited dilution PCR

• Each individual reaction either contains a DNA molecule or not, gives yes or no signal, some may contain multiple 

• At low lambda values most reactions contain no target molecules and few a single copy

• At high lambda values nearly all reactions are positive with multiple copies of the target molecule

Advantages and disadvantages of dPCR

• Advantages - absolute quantification with no standard curve, very sensitive, increased resistance to inhibitors, fast TAT (2h)

• Disadvantages - lower dynamic range, some samples may need further dilution if initial input was high

Digital PCR platforms

• Plate based systems - sample dilutes, partitioned into thousands of individual reactions, end-point PCR performed on partitions, readout and absolute quantification

• Emulsion based systems - reaction chambers consist of small water droplets separated by oil (droplet digital PCR)

Data visualisation

• 1D plots - one axis, relative fluorescence units (RFU), any dots above threshold are positive partitions

• 2D plots - multiplexing 2 reactions, RFU on both axis (different colours), negative in bottom left, double positive in upper right

Secondary analysis

• Based on results of the absolute quantification

• Mutation detection, copy number analysis, gene expression analysis

Applications of PCR assays

• Microbiology - pathogen detection, design primers in highly conserved regions due to high mutations, load can be determines

• SNV - one set of primers with two allele specific fluorescent labelled probes used e.g. FVL mutation

• Foetal RHD genotyping - cell free foetal DNA in maternal circulation, qPCR used to amplify RHD gene

• Measurable residual disease - qPCR for detection of MRD

• Chromosomal translocations - tumour specific PCR targets, primers designed to anneal to opposite sides of the breakpoint