PCR and it's role in diagnostics

Question 1

Define the polymerase chain reaction

Answer 1

PCR: 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? (2)

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

Question 3

What condition makes PCR specific and what does this prevent?

Answer 3

• Is specific only if annealing is undertaken at the melting temperature Tm of the primers, ie high stringency conditions
• This prevents mis-matched based pairing

Question 4

The segment being amplified is determined by the end sequences… what does this therefore mean?

Answer 4

• If we want to amplify a segment bounded by known sequence we can do this by choosing primers complementary to these ends and exponential amplification requires two primers each complemeary

Question 5

What does DNA dependant DNA Polymerase do?

Answer 5

• The enzyme recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it.

Question 6

Describe the first stage of PCR (4)

After the DNA dependant polymerase has formed an initiation complex

What is the newly formed strand called?

Answer 6

• In PCR a partially double stranded structure is formed by annealing a short single stranded DNA molecule (a primer)
• In order to achieve this, the double stranded template has first to be denatured and thus made into single stranded molecule
• It is performed only after the template is denatured by heat
• The newly formed strand is sometimes referred to as the nascent strand

Question 7

What can annealing also be called?

How are high stringency conditions achieved during annealing

What does annealing result in?

Answer 7

• Annealing is an alternative way of describing hybridisation
• Annealing of the primer under high stringency conditions is achieved using the predicted melting temperature of the primer-template duplex.
• Annealing results from the formation of base-pairing, stabilised by hydrogen bonding

Question 8

Is annealing of the two primers to each of the separate template strands and renaturation a competitive process?

Answer 8

Yes

Question 9

What is the formation of the primer-template complex driven by?

What effect does this have on the equilibrium?

Answer 9

• The formation of primer-template duplex is driven by favourable kinetics provided by a huge molar excess of the primer
• Thus the equilibrium due to competition between renaturation of the double stranded template and annealing of the primer to the template preferentially occurs towards the primer template annealing

Question 10

What are the 3 things we need for PCR?

Answer 10

1. A template strand with a primer (usually 20-30 bases long) annealed to it with a 3 prime OH group and an 5 primer overhanging template strand
2. Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP) to form the elongating strand, the consequence of incorporating these in to the elongating strand is that we hydrolyse the triphosphate adding a mono-phostphate the strand and release pyrophosphate and hydrogen ions. This leads to an acidification of the reaction and depletion of reactants
3. In your first year we talked about cofactors and Mg2+ ions are required as an essential cofactor for all DNA polymerases and if we remove or chelate Magnesium, we will inhibit the reaction

Question 11

PCR transitions through 3 different states, what are these states?

Answer 11

• Denatured - where the template is single stranded where the template has been heated and the hydrogen bonds stabilising the duplex broken, this also means we need to use an enzyme that is capable of withstanding the harsh conditions used
• Annealed - the formation of a duplex between the primers and corresponding template strands
• The Native state – this is where optimal conditions for the extension of the initiation complex and enzyme activity occurs

Question 12

For PCR to work the reaction MUST go through multiple rounds of extreme heating and cooling, most enzymes would not tolerate such conditions.

What is a condition that the taq polyemrase enzyme must-have?

Define thermostability

Where is this enzyme obtained from?

Answer 12

• so the polymerase MUST be thermostable
• Thermostability means the enzyme is “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 13

Briefly give a quick overview of the PCR process

Answer 13

• Firstly we need to assemble the reaction components : template , enzyme cofactors, buffer and other reactants
• The first stage is denaturation where we heat the reaction to temperatures in excess of 95 degrees,
• followed by cooling to the tm of the primer –template duplex followed by adjusting these conditions to the optimal conditions for the enzyme to elongate the first round product which the can act as template in subsequent rounds
30 cycles of PCR will amplify a single molecule 1 billion times so an incredibly powerful technique

• Every cycle results in a doubling of the amount of product, thus is an exponential accumulation of product
• The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction

Question 14

How is PCR used in diagnotsics?

Give some examples

Answer 14

• routine diagnostic tool used for identification, confirmation and quantification of specific DNA sequence

• 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 15

We’ve seen that in PCR, the amplification follows a sigmoidal curve and the reaction becomes inhibited as it progresses. explain this

Answer 15

This is as a result of the reactants becoming depleted or the reaction itself being poisoned by acidification.

Question 16

Explain the plateu of the number of PCR molecules after every cycle

Answer 16

As we can see from the graph, the amount of product at the end of the reaction does not reflect the amount of template at the start ,since we have a plateauing of the amount of product.
• In the graph each line represents different amounts of starting material and all plateau at the same level or end point as the reaction becomes rate limited
• This means, that this form of PCR does not have a quantitative output and the end point of the reaction cannot be used to inform template copy number
• But what we can see is that as starting copy number increases, the curve shifts to the left and so the position of the curve does reflect the template copy number at the start.
• We can use this characteristic if we measure the increase in yield during the reaction in real-time

Question 17

What is qPCR?

What does it allow us to monitor?

Answer 17

These techniques use fluorescent detection of the accumulating product at the end of each cycle of PCR
This allows us to monitor the early part of the reaction,
• the point at which product becomes detectible, reflects the starting quantity in the reaction
• if we place a threshold on the measurement of product and compare this to a standard curve of known concentration this 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 18

What are SNPS?

Answer 18

Single Nucleotide Polymorphisms or SNPs, these single base variations are used as genetic markers for association studies amongst other things, but rare single base variants can also be deleterious for example causing sickle cell Anaemia

Question 19

Describe common Applications for detecting single nucleotide polymorphisms or mutations

Answer 19

• Antibiotic resistance testing –for example fluoroquinolone resistance in TB conferred by gyrA mutations and many other organisms
• Another example might be the Identification of genetic markers – for example
• Those conferring drug sensitivity or catabolism (for example CYP2C9 and VKORC1 variants confer warfarin sensitivity),
• markers of disease (such as BRCA1 & 2 mutations in breast cancer)
• or treatment response (for example efficacy of ribavirin in treatment of Hepatitis C virus , determined by IL27 variants)

Question 20

What are the two two most common methodologies that we might use to determine the presence of SNPs ?

Answer 20

HIGH RESOLUTION MELTING and ALLELIC DISCRIMINATION

Question 21

Describe the premise of high resolution melting

Answer 21

• So HIGH RESOLUTION MELTING relies upon the effect that differences in the amplified product containing a variant has on the melting curve for a given sequence.

Question 22

Describe the premise of ALLELIC DISCRIMINATION

Answer 22

• The second method ALLELIC DISCRIMINATION relies upon short probes that are internal to the flanking primers used during qPCR, and that span the SNP and allows the determination of the presence or absence of the SNP.

This is  due to differences in the Tm caused by matching and mis-matched base pairing of the probe.  •	An example of this can be seen on the left where  the T homozygote (orange circles ) are shown above the blue line,  the c homozygote (blue circles) to the right of the orange line and the  heterozygotes (green triangles) are shown on the diagonal

Question 23

How is PCR used in forensics and law enforcement amplification which uses other types of genetic markers that might indicate….. (4)

Answer 23

• For example Parentage or kinship: and this might be used in immigration and inheritance
• We might us this approach for the Identification of an individual : for example military casualties, missing persons or in the case of environmental disasters
• We can of course also use the technique for matching individuals with biological samples obtained from crime scenes
• We can also use PCR for the Authentication of biological materials: cell lines or the purity and origin of foods (for example meat products)

Question 24

What are STR’s?

Are they polymorphic?

What do they provide?

Answer 24

Forensics Short tandem repeats

• 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
• Provide a pattern of uniquely sized products accorded by each individuals genome
STRs thus form a type of molecular bar code specific to each individual and are akin to a finger print

Question 25

Using STRs how can we identify people?

Answer 25

• The technique uses PCR combined with labelled primers that are designed to flank and thus amplify each STR. As indicated at the top of the slide where the primers are represented by the arrows and the block of colour in the middle of the bar, the STR.
• The products of the PCR reaction are separated on a capillary gel according to their size
• The label identifies which of the 10 STRs it represents, along with the number of repeats at each allele.
• You can see such a readout of the electrophoresis in the image,
if we take the STR at locus 1, VWA we can see two peaks representing the two alleles each comprising of 14 and 16 repeats respectively
In the electropherogram if the alleles are of different size the would show as two peaks and if they were if the same size as with locus 2 only a single peak would be present

Question 26

What are the other applications of PCR?

Answer 26

Amplifying material prior to:
• Next generation sequencing eg. Simultaneously sequencing large number multiple PCR products of candidate Cancer genes
• Isolating individual segments of DNA prior to cloning or sequencing

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

PCR is one of the most commonly used and important tools used in Recombinant DNA technology
• eg developing recombinant vaccines, pharmaceuticals (interferons, clotting factors, Tissue plasminogen Activator etc)