PCR & its role in diagnostics

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction: is an enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process.

Question 2

What is a chain reaction?

Answer 2

A Chain reaction is a series of events each one of which is dependant upon the preceding event to sustain itself.

Typically it is a series of reactions that lead to an exponential increase in the number of events occurring in a sequence.

In PCR there is the doubling of the number molecules produced through the each stage of the amplification,
and the product of one cycle provides the template for the subsequent cycles.

So we start off with an one double stranded molecule of DNA, after 2 cycles –> 4 molecules, 4 cycles 16 molecules and 6 cycles 64 molecules - an exponential increase in molecules until the reaction is exhausted so there’s an exponent to the base 2 and after n cycles we’d have 2’n number of molecules produce from a single DNA molecule.

Question 3

Under which conditions is PCR specific?

Answer 3

PCR is a method to specifically amplify segments of DNA. It is specific only if annealing is undertaken at the melting temperature (Tm) of the primers (ie. high stringency conditions). This prevents mismatched based pairing.

Question 4

What is the amplicon and how is it determined?

Answer 4

The amplicon is a piece of DNA/RNA that is the source and/or product of amplication or replication events (chain reaction).

After the DNA is denatured and the strands separated. The segment amplified is called the amplicon and is determined by the sequence at the ends of that section of DNA.

Question 5

How is specificity determined in PCR?

Answer 5

Specificity is determined in PCR by making oligonucleotides (short DNA or RNA molecules) called primers which are complementary to the sequences at the ends of the amplicon and form a duplex by hybridising to them. DNA polymerase recognises these duplexes and forms an initiation complex around them

Specificity is thus determined by the uniqueness of these sequences and their complementarity.

Hybridising of primers is referred to as annealing primers and the temperature we use to hybridise them, the Tm of the duplex, is referred to as the annealing temperature.

Question 6

How does exponential amplification occur?

Answer 6

The portion of the DNA amplified is determined by the choice of primers and their complementarity to the ends of the amplicon. The exponential amplification requires two primers, each complementary to one of the two strands.

The polymerase is then able to form initiation complexes and make new strands in the direction that produces the products that are overlapping and complementary.

Question 7

What is a DNA-dependant DNA Polymerase, and what are its functions?

Answer 7

It is the enzyme used in PCR that 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 (ie. adds nucleotides to the 3’ end of the priming strand).

Question 8

What is annealing, and how does it play a role in PCR?

Answer 8

In PCR, a partially double stranded structure with a 3’ end, and an over-hanging 5’ template strand, is formed by annealing (hybridising) a primer (short ssDNA molecule) to the template strand

To achieve this, the double stranded template is denatured - separating the two strands. Thus forming two single stranded molecules. This is achieved by heating the reaction to a temperature that breaks the H-bonds stabilising the duplex.

The newly formed strand is referred to as the nascent strand.

As a consequence, we’re forming a partially double-stranded molecule that can then act as a template for PCR.

Question 9

How does the primer anneal to the template strand?

Answer 9

Annealing is an alternate way of describing hybridisation. Annealing of the primer under high stringent conditions is achieved using the predicted melting temperature of the primer template duplex. It results from the formation of base pairing, stabilised by H-bonds. It relies on the complementarity of the primer and the target molecule, thus forming a duplex with specific perfectly matching base pairs.

Question 10

How does the annealing of the primer compete with the renaturation of the template?

Answer 10

The template at the start of the reaction is in a low concentration. The formation of the primer template duplex is forced to occur by providing a huge excess of the primer.

This is in a competition between the renaturation of the double-stranded template and the annealing of the primer to the template, in which the annealing is in preference to renaturation is driven by favourable kinetics as a result of the vast excess of the primer present in the reaction.

Question 11

What is meant when a DNA-dependant DNA Polymerase is used?

Answer 11

• it synthesises a new nucleic acid strand by copying a DNA molecule
• it cannot copy RNA nor make RNA
• RNA must first be copied to complemtaty DNA (cDNA) by reverse transcription before it can be amplified by PCR.

Question 12

What are the requirements of the PCR?

Answer 12

1. A template strand with a primer (usually 20-30 bases long) annealed to it with a 3 prime OH group and a 5 primer overhanging template strand.
2. Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP) to form the elongating strand. (Incorporating these in the elongating strand leads to hydrolysing the triphosphate, adding a mono-phostphate to the strand and releasing pyrophosphate and hydrogen ions. Leading to an acidification of the reaction and depletion of reactants)
3. Mg2+ ions - essential cofactor for all DNA polymerases. Removing or chelating Magnesium will inhibit reaction.
4. A roughly neutral pH - required for buffering since the reaction itself produces H+ ions.

Question 13

What are the 3 transition states of PCR and what are they reliant upon?

Answer 13

PCR is a cyclical process and is based on transitions between 3 states reliant on hybridisation of primers and formation of partial duplexes. The 3 states are:

• Denatured state (template becomes single stranded using heat and enzyme)
• Annealed state (formation of duplex with the primer and initial template strand)
• Native state at the optimal extension temperature and pH for enzyme activity.

Question 14

Why is thermostability important in PCR?

Answer 14

For PCR to work, the reaction must go through multiple rounds of extreme heating and cooling, so the polymerase must be thermostable.

Thermostability means ‘able to retain activity’ upon repeated heating to temperatures that would “destroy” most enzymes.

Hence, a polymerase from a thermophilic bacterium such as Thermus Aquaticus is often used (Taq Polymerase).

Question 15

Briefly, what are the three steps of PCR?

Answer 15

Assemble reaction components and mix (template strand, primers, enzyme, and reactants) in a test tube.

1) we denature them at 95°C
2) we then anneal at the Tm of the primers at 55°C
3) extend from the 3’ end of the primers at 72°C

30 cycles = 1 billion copies

Each cycle = doubling of the amount of product = exponential accumulation of product.

However the reaction kinetics are determined by:

• Depletion of reactants
• Acidification of the reaction due to H+ ions produced during elongation and is seen by the plateauing of the graph.

Question 16

How can PCR be used in diagnostics and give some examples?

Answer 16

PCR is a routine diagnostic tool used for identification, confirmation and quantification of specific DNA sequence.

Examples:

• Presence absence calling TB - detection in sputum, determining treatment choice.
• Differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes.
• How much is present - determining when treatment might be commenced, “HIV viral load”.
• Identifying individuals positive for SARS-CoV-2 and thus have CoVID-19.

Question 17

How is PCR qualitative and not quantitative?

Answer 17

The end of the PCR reaction does not have a quantitative output and cannot be used to inform template copy number.

Regardless of the starting concentration of template (its independent of the starting concentration), the same endpoint is reached as amplification becomes rate limited.

Thus PCR is qualitative in the sense that it can determine the presence or absence of a substance.

Question 18

Why do we use modifications to the PCR technique?

Answer 18

We use modifications of the technique to provide measurable output during the exponential phase of the amplification in ‘real-time’.

Question 19

Explain how PCR is made quantitative?

Answer 19

There are a number of different quantitative PCR detection methods used for diagnostics. Collectively they’re referred to as real-time PCR or quantitative PCR (qPCR).

These techniques utilize fluorescent detection of the amplification. They’re used for quantifying the amount of a target DNA molecule in the sample.

This allows early monitoring of the reaction - the crossing point at which product becomes detectible (is determined and proportional to the template concentration), reflects the starting quantity in the reaction.

Placing a threshold measurement of the product and comparing this to a standard curve of known concentration makes the technique quantitative.

This approach forms the basis of most diagnostic assays today, including detection and amount of coronavirus an individual may have.

Question 20

What are SNPs?

Answer 20

A single nucleotide polymorphism (SNP) is the most common type of genetic variation among people. Each SNP represents a difference in a single DNA building block, called a nucleotide. Most commonly, these variations are found in the DNA between genes.

Question 21

How can PCR be used for SNP detection?

Answer 21

PCR can be used to detect Single nucleotide polymorphisms (SNPs) and single base alterations – single nucleotide genetic variants.

Several methodologies enable us to detect single nucleotide genetic variants - 2 are adaptations of quantitative real-time PCR which allow the detection of SNPs or single base variants such as cancer mutations or drug resistance mutations.

These methods rely on the differences in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition (duplex containing a single nucleotide mismatch).

• rare single base variants can be deleterious, causing sickle cell Anaemia for example.

Question 22

What are some common applications of SNP or mutation detection?

Answer 22

Common Applications:
- Antibiotic resistance testing e.g. fluoroquinolone resistance in TB conferred by gyr A and many other organisms.

• Identification of genetic markers - drug sensitivity/catabolism (CYP2C9 and VKORC1 variants confer warfarin sensitivity), markers of disease (such as BRA 1&2 cancer) or treatment response (HCV determined by IL27 variants).

Question 23

What are the two approaches to SNP detection, specifically?

Answer 23

• High resolution melting (HRM): the Tm of the amplified product is used to determine which sequence is present. Variation is seen as a result from SNPs being present in different amplicons
• Probe based version of qPCR (sometimes referred to as Allelic discrimination): where specific binding of the probe to the amplified region containing the SNP is detected. This is due to differences in the Tm caused by matching and mis-matched base pairing of the probe.

Question 24

What are the applications of PCR in forensics and law enforcement?

Answer 24

• 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
• Authentification of biological material: cell lines, purity of foods

Question 25

Describe forensic short tandem repeats (STRs or microsatellites).

Answer 25

Forensic identification uses repetitive sequences (STRs)

STRs are 2-5 or more bases in length, repeated many times at specific locations in the genome. Many different STRs are found scattered around the genome. They are highly polymorphic (ie. the number of repeats varies between individuals).

They provide a pattern of uniquely sized products, accorded by each individuals genome, providing a molecular barcode or ‘DNA fingerprint’.

The UK DNA database currently consists of 10 STRs, each differing in size; giving 20 numbers and a gender indicator. This gives a matching probability with an error of around 1 in 1 billion.

Question 26

How do STRs work?

Answer 26

The technique uses PCR combined with 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.

The products of the PCR reaction are separated on a capillary gel according to their size and can be seen on an electropherogram.

Question 27

What are some other applications of PCR?

Answer 27

Amplifying material prior to:

• Next generation sequencing, NGS, (eg. simultaneously sequencing large number multiple PCR products of candidate cancer genes)
• Isolating individual segments of DNA prior to cloning or sequencing (e.g. from biopsies, blood samples)
• Manipulating and modifying DNA: Introducing mutations into a sequence of DNA. Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors.
• Used in recombinant DNA technology eg. in developing recombinant vaccines, pharmaceuticals (interferons, clotting factors, tissue plasminogen activator etc).