Polymerase chain reaction

Question 1

Define polymerase chain reaction.

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

Define 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

Question 3

What conditions does PCR need to be performed under?

Answer 3

It 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

How is a specific known segment amplified?

Answer 4

Since the segment amplified is determined by the sequence at the ends
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 is the polymerase that is used in PCR?

Answer 5

DNA dependant DNA polymerase

Question 6

How is the polymerase used?

Answer 6

The enzyme 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

Question 7

How is a partially double stranded structure formed in PCR?

Answer 7

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 8

What is annealing and how is it used in PCR?

Answer 8

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 9

What is the relationship between annealing of the primer and renaturation of the template?

Answer 9

Annealing & renaturation are a competitive processes
Annealing of the primer occurs in preference to renaturation and is driven by favourable kinetics as a result of the vast excess of the primer present in the reaction

Question 10

Give some rules that come with the use of using DNA dependant DNA polymerase in PCR.

Answer 10

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

Question 11

What does PCR require fr the reaction to occur?

Answer 11

• A template strand with a primer (usually 20-30 bases long) annealed to it
• Deoxy nucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
• Mg2+ ions
• A roughly neutral pH

Question 12

What are the 3 states of PCR?

Answer 12

• Denatured (template becomes single stranded)
• Annealed (formation of initiating template)
• Native state at the optimal extension temperature and pH for enzyme activity

Question 13

What are the rules of PCR when it comes to temperture?

Answer 13

-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 (eg Taq polymerase)

Question 14

What are the steps for PCR?

Answer 14

1. The components for PCR are assembled. (Template, Primers, Enzyme & Reactants)
2. Denaturation of the template by heating of the mixture to 95 degrees C
3. Then cooled to the Tm of the primer-template duplex which is 55 degrees
4. Then heated again to 72 degrees for the formation of the initiation complex which will then allow the extension of the copy strand.
5. this process is repeated 30-40 times to produce 1 billion copies.

Question 15

Why does the end of PCR not have a quantitative output?

Answer 15

Because different starting template concentrations all have the same endpoint due to the reaction (amplification) becoming rate limited

*Therefore, the end of the PCR reaction cannot be used to determine the starting concentration of the template

Question 16

What is Real-time PCR (qPCR)?

Answer 16

PCR based method which is used to amplify and quantify a targeted DNA molecule. Enables both detection and quantification.

Provides measurable output during the exponential phase of the amplification in ‘real time’

Question 17

How does real-time PCR work?

Answer 17

Fluorescent molecule binds product of amplification and fluoresces, meaning we can quantify the amount of material being produced by the amount of fluorescence produced through the specific binding of that molecule the product.

The more product we accumulate, the more fluorescence we gain, meaning the greater the starting concentration is

Question 18

What are the steps to real time PCR?

Answer 18

1. Perform PCR on a sample and at same time perform a standard curve with known concentration of template and a fluorescence molecule in the mixture
2. Place a fluorescence threshold across those curves
3. Point at which the fluorescence crosses that flurorescence detection threshold reflects the starting concentration fo material put in a PCR reaction. This crossing point is proportional to the template concentration at the start

> the quicker it takes a DNA sample to cross the fluorescence threshold level, the more abundant it is

Question 19

What are the applications of PCR?

Answer 19

• Diagnostics
• SNP Detection
• Forensics and Law Enforcement
• Recombinant DNA Technology

Question 20

How is PCR used in diagnostics?

Answer 20

Used for identification, confirmation and quantification of specific DNA sequence:

• presence/absence of TB
• differentiating between closely related organisms e.g. swine flu vs human influenza (both H1N1 subtypes)
• amount of molecule present in sample to determine when treatment might be commenced e.g. HIV viral load in individual blood

Question 21

How is PCR used in SNP detection?

Answer 21

Used to determine and differentiate between SNPs within a population of molecules:

• Antibiotic Resistance Testing: TB and other organisms
• Identification of Genetic Marker: drug sensitivity/catabolism (CYP2C9 and VKORC1 variants confer warfarin sensitivity), markers of disease (cancer) or treatment response (HCV)

There are 2 approaches in detecting SNPs:

1) High Resolution Melting (HRM)- difference in the Tm of the amplified product is used to determine presence of SNP
2) Probe Based Version of qPCR (Allelic Discrimination)- specific binding of the probe to the amplified region containing SNP is detected

Question 22

How is PCR used in forensics and law enforcement?

Answer 22

Forensic Identification uses STRs, which are 2-5 bases in length repeated many times at specific locations in the genome and are highly polymorphic (number of repeats varies between individuals)

Multiple labelled primers flank different STRs (short tandem repeats) and the products are separated on a gel by size and the label is read to identify the size of the STR and which one of 10 reference STRs in UK database it represents

Used in amplification of genetic markers for:

• Parentage or Kinship: immigrations and inheritance
• Identification: military casualties, missing person or environmental disasters
• Matching two sources: crime scene
• Authentification of biological material: cell lines, purity of foods

Question 23

How is PCR used in recombinant DNA technology?

Answer 23

One of the most commonly used tools and important tools in Recombinant DNA Technology by the pharmaceutical industry
>used to develop recombinant vaccines, pharmaceuticals (interferons, clotting factors, tPA etc)