PCR

What are the different types of PCR?

• conventional

• using random hexamers

• hot start

• bidirectional allele specific PCR

• reverse transcriptase PCR

• multiplex PCR

• qRT-PCR

• droplet digital PCR

What is a conventional PCR reaction mix?

• DNA template

• dNTPs

• primers

• plymerase

• reaction buffer

What is the most important component of PCR reaction buffer and why?

MgCl₂ as it is required for the activity of the polymerase (varying its concentration controls the efficiency of the reaction)

What are the steps in a basic PCR reaction?

1) denaturation (30 sec - 1 min at 94 °C) - separation of the two strands of template DNA

2) annealing (30 sec - 1 min at 55-60 °C) - primers bind to the separated template strands

3) extension (1 min per Kd at 72 °C) - synthesis of new strands

What are the advantages of using PCR for DNA amplification?

• huge capacity for amplification

• high specificity

• entirely in vitro

• efficient

• sensitive (from a single copy)

• rapid

• cheap

• don’t necessarily need to know the entire sequence

What are the main applications of PCR?

1) research

• DNA amplification

• conversion of RNA to cDNA

• introducing sticky ends for gene cloning

• introducing SNPs (site-directed mutagenesis)

• gene expression analysis (qRT-PCR)

• mutation detection

2) screening/diagnostic

• detection of infectious diseases (e.g., HIV, COVID-19, tuberculosis) by identifying pathogen DNA/RNA

• genetic disorder screening, including inherited mutations (e.g., cystic fibrosis, sickle cell anemia)

3) Forensic science

• DNA fingerprinting

• Paternity testing

How is PCR using for DNA fingerprinting?

• DNA is extracted from biological samples

• primers are designed to target short tandem repeats (STRs) or variable number tandem repeats (VNTRs), which are regions that differ in length and sequence among individuals

• PCR amplifies these regions

• The amplified DNA fragments are separated by gel electrophoresis

• Fragment sizes are measured and visualized as a pattern of bands

• The DNA fingerprint (band pattern) from a sample is compared with a suspect’s DNA, a victim’s DNA, a database profile

• A match across multiple STR loci provides extremely high confidence of identity

How is PCR used for paternity testing?

• DNA is isolated from each sample

• Primers are designed to target short tandem repeats (STRs), which are regions that differ in length and sequence among individuals. Each person has two alleles per STR locus, one inherited from each parent.

• PCR amplifies these regions

• Amplified STR fragments are separated. The length of each fragment corresponds to the number of repeats, producing a DNA profile

• The child’s STR alleles are compared with those of the mother and alleged father. At each locus: one allele must match the mother, the other must match the biological father.

What are the two primers required for PCR?

• forward primer (binds to the 3′ end of the antisense (template) strand)

• reverse primer (binds to the 3′ end of the sense (coding) strand)

What are the considerations for PCR primer design?

• they need to flank the DNA sequence to be amplified

• 17-30 bp long (to be specific to the gene of interest)

• >5% GC content (for tight binding to the template)

• no complementarity between primers (to avoid primer dimers)

• no secondary structures (e.g. hairpins) within primers

How can PCR product be visualised?

by running it on agarose gel (DNA is stained with ethidium bromide, so can be visualised under UV light)

How many times is the PCR cycle repeated?

30-40

What is the purpose of PCR with random hexers and how does it work?

When you don’t know the sequence of the template (so can’t design specific primers), short segments (6-8bp) of single-stranded DNA with all possible combinations of the dNTPs are used.

What are the issues with conventional PCR?

• contamination (with exogenous DNA)

• non-quantitative

What are the possible reason for a PCR reaction not working?

• 90% due to primers (non-specific binding, primer dimers)

• contamination

• template degradation

• enzyme denaturation

What kind of DNA polymerases are used for PCR reactions?

ones that can withstand high temperatures as DNA is very stable and only denatures at 94 °C (most proteins denature at 70 °C, so polymerases from bacteria that live in hot environments are used)

What are examples of polymerases used for PCR?

Taq, Pfu, KOD Hot start

What are the characteristics of Taq polymerase?

quick, cheap, but has a high mutation frequency

What are the characteristics of Pfu polymerase?

relatively slow, but has DNA proof-reading mechanisms so lower mutation frequency

What is the purpose of Hot start PCR and how does it work?

reduces non-specific amplification and formation of primer dimers (during the experimental setup the polymerase is blocked by antibodies as this is the step when those issues mainly take place, then a heating stage is added at the beginning of the reaction to denature the antibodies and activate the polymerase)

What is the purpose of bidirectional allele specific PCR and how does it work?

allows to detect polymorphisms-SNPs

• two sets of primers are designed (one for WT, one for mutant)

• one primer of each set is complimentary to SNP at the 3’ end

• each pairs with a different “outer” primer, resulting in PCR products of different sizes, depending on the alleles

• if both hands can be seen - heterozygous

What is the purpose of reverse transcriptase PCR and how does it work?

used to produce a cDNA sequence from RNA, so is can be expressed

What is the purpose of multiplex PCR and how does it work?

increases efficiency as can amplify several different DNA segments as the same time (from a single or multiple templates)

• multiple pairs of primers are designed

What are the benefits of multiplex PCR?

• increases efficiency as can amplify several different DNA segments as the same time

• gives some indication of product quantity by comparing products to each other

What are the challenges of multiplex PCR?

• careful primer optimisation required (to make sure they are very specific and have similar annealing temperatures)

• need to amplify a standard housekeeping gene as an internal control

What is the purpose of qRT-PCR and how does it work?

allows to calculate the amount of DNA amplification started from

• a fluorescent molecule that binds to DNA is added to the reaction

• when DNA is denatured, there in no fluorescent signal

• as the double-stranded DNA starts to form, fluorescence increases

• the reaction is followed in real time, the fluorescent signal increases proportionally with DNA amplification

What are the two main detection methods for qRT-PCR and how do they work?

1) SYBR Green

• it is a dye that binds to double-stranded DNA, and it fluoresces when it is bound

2) molecular probe

• a PCR primer combined with a fluorophore and a quencher (when unbound, quencher blocks the fluorophore, when primer binds, quencher is separated from fluorophore)

What are the advantages of using a molecular probe for qRT-PCR?

• higher specificity

• enables single tube multiplexing of qRT-PCR reactions for multiple targets

How are data for qRT-PCR interpreted?

• a standard curve can be created using known amounts of template and then used to determine the concentration of a test template

• a reference (housekeeping gene) can be used for relatively quantification (reported as fold increase)

What are the main challenges of PCR?

• primer design (melting temperature, complementarity, secondary structure, amplicon size, spanning at least one exon-exon junction to prevent amplification of contaminating DNA)

• RNA being easily degraded

What controls are required for a PCR reaction?

• no template control (to check for contamination)

• no amplification control (without enzyme)

• referense control (e.g 18S RNA)

How can contamination during PCR be reduced?

• using master mixes

• using primers that span at least one exon-exon junction)

• using hot start neutralising antibodies

What is the purpose of droplet digital PCR and how does it work?

allows precise, highly sensitive absolute quantification of DNA

• sample is diluted to approximately 1 copy template per well

• PCR reaction is carried out on “individual” copies

• presence of product is detected by fluorescent probe (positive and negative wells depending on presence of template)

• Poisson distribution analysis is used to calculate absolute quantity

How can PCR be used to detect infectious diseases?

multiplex qRT-PCR can be used to identify variants of COVID by designing primers to amplify specific genetic sequences that belong to a particular strain

viral load can also be calculated

How can genetic disorders be identified using PCR?

• allele specific PCR can detect point mutations (by designing primers that one allow amplification when a specific nucleotide is present)

• ddPCR (droplet digital PCR) is used for detection of rare mutations present at very low percentages or circulation cancer DNA from liquid biopsies)