PCR and its roles in diagnostics

Question 1

What is the polymerase chain reaction (PCR)?

Answer 1

The polymerase Chain Reaction (PCR) is an enzyme-based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process.

Question 2

What 2 aspects of PCR control the specificity of the technique?

Answer 2

• The complementarity of the primers to the specific sequence of DNA you want to amplify
• The uniqueness of the primer sequences used.

Question 3

Why is the specificity of PCR dependent on the complementarity of the primers to the target DNA sequence?

Answer 3

Because if the primers used weren’t complementary to the target DNA sequence you wouldn’t be able to amplify the target sequence because they wouldn’t be able to bind meaning DNA polymerase wouldn’t have a template to use to amplify the target sequence.

Question 4

Why is the specificity of PCR also dependent on the uniqueness of the primers used?

Answer 4

Because if the sequence of the primers used is not unique then you get complementary to lots of different sequences within the DNA sample which means you will amplify loads of different sequences of DNA.

Question 5

How is the target sequence for PCR identified?

Answer 5

Within the target molecule you identify a sequence that flanks the sequence you want to amplify - this sequence is called the flanking sequence

Within the flanking sequence you then identify a segment of DNA that has the same T_m as that of the primers.

Question 6

Within the target DNA sequence how do the primers orientate themselves and why is this the case?

Answer 6

The primers are oriented in opposite directions to each other so that the DNA polymerase moves towards the opposing primer as it amplifies one of the primer sequences.

Question 7

What type of DNA polyermase is used during PCR?

Answer 7

DNA-dependent DNA polyermase

Question 8

In PCR once the DNA duplex has been denatured what are the 2 possibilties that can happen? Which one of these 2 processes is favoured during PCR

Answer 8

• Annealing of the primers to the template strand
• Renaturation of the 2 denatured strands to reform the DNA duplex
• Annelaing of the primers to the template starnd is favoured during PCR

Question 9

Why is the anneling of the primers to the template strand favoured over the renaturation of the 2 denaturated strands after the DNA has been denatured during PCR?

Answer 9

Because you have a high molar excess of the primer within the reaction mixture compared to the original strand of the duplex. This means there is a much higher chance of the primer forming hydrogen bonds with the template strand then there is the original strand of DNA reforming hydrogen bonds with the template strand and reforming the original duplex.

Question 10

What conditions are needed for the DNA-dependent DNA polymerase to be able to synthesise the non-template strand?

Answer 10

• A template strand with a primer (usually 20-30 bases long) annealed to it
• Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP) to form the elongating strand
• Mg2+ ions (required as a cofactor for the enzyme)
• Roughly neutral pH

Question 11

PCR involves the transition between 3 different states, what are they?

Answer 11

• Denatured - where the DNA duplex becomes single stranded
• Annealed - reaction mixture cooled which allows for the formation of a partial duplex with the primer and template strand
• Native state – optimal conditions for the DNA-dependent DNA polymerase to form an initiation complex with the partial duplex and then extend the elongate the non-template strand.

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Question 12

Why must the DNA-dependent DNA polymerase have high thermostability?

Answer 12

For PCR to work the reaction must go through multiple rounds of extreme heating and cooling which means the DNA-dependent polymerase must be able to retain activity even at temperatures that would denature most enzymes.

Question 13

Describe the steps involved in the polymerase chain reaction?

Answer 13

1. Add into reaction mixture DNA template, DNA dependent DNA polymerase, 2 oppositely oriented primers and other reactants (Mg2+ ions, deoxyribonucleoside triphosphates, dNTPs, and a buffer).
2. Raise the temperature of the reaction mixture in order to break hydrogen bonds within the DNA duplex causing it to denature and form 2 single strands.
3. Cool the reaction mixture to temperature of the T_m of the primers allowing them to anneal to template strand and form a partial duplex.
4. Change the temperature again so now it is at the optimal temperature for the DNA-dependent DNA polymerase which results in it forming an initiation complex with the partial duplex. It then goes on to efficiently elongate the non-template strand.
5. Repeat these steps multiple times to produce millions of copies of the amplified section of DNA - PCR usually involves around 30 to 40 cycles.

Question 14

What 2 factors affect the kinetics of the polyermase chain reaction?

Answer 14

• The depletion of reactants - removal dTNPs from reaction mixture as well as incorporating primers into the elongated strand
• The acidification of the reaction

Question 15

How does the reaction mixture become more acidic over the course of a PCR recation?

Answer 15

As the DNA-dependent DNA polymerase adds deoxyribonucleoside triphosphates to the non-template strand they become monophosphates and as a result pyrophosphate and H⁺ ions are produced. The accumulation of H⁺ ions eventually overcomes the effect of the buffer within the reaction mixture causing it to acidify.

Question 16

Why does acidification of the reaction mixture affect the kinetics of the PCR recation?

Answer 16

Acidification of the reaction mixture results in the inhibition of the DNA-dependent DNA polymerase which means the reaction will not be able to continue.

Question 17

What are some common diagnostic applications of PCR?

Answer 17

• Used to identify presence/absence of infectious organisms such as mycobacterium tuberculosis within a person - detected by taking sputum sample
• Used to determine no. of molecules present within a sample - used to determine how many HIV particles are present within the blood sample of an individual (Viral load test).

Question 18

Why is standard PCR not classed as a quantitative technique?

Answer 18

Because no matter what the starting concentration of DNA is the reaction will always have the same end-point. This means that the end-point in PCR can not be used to determine the starting concentration of the DNA which means standard PCR isn’t a quantative method.

Question 19

How do you make PCR a quantitative technique?

Answer 19

• You add a fluorescent probe to the reaction mixture which is capable of binding to the product of PCR.
• You then use the results of the PCR reaction to plot a graph of fluoresence against cycle number
• On this graph you plot the threshold fluoresence (C_T threshold) and it to find the point at which the fluoresence for each DNA smaple crosses the threshold fluoresence (C_T value)
• You then use this C_T value to work out which sample had the highest starting concentration - the lower the C_T value the higer the starting concentration

Question 20

What is the name of the PCR technique that can be used as a quantatitive method?

Answer 20

Real-time PCR or Quantitative PCR (qPCR)

Question 21

What are the 2 PCR-based methods used to detect SNPs?

Answer 21

• High resolution melting - where the T_m of the amplified product (amplicon) is used to determine the presence of a SNP. The T_m and the characteristic melting curve of the associated with the amplicon will be different due to the presence of any SNPs.
• Allelic discrimination - where specicfic binding of a short probe spanning the SNP is used during qPCR. The binding of the short probe will give the amplicon a different T_m which is then detected.

Question 22

What does PCR SNP detection depend on?

Answer 22

Use of PCR to detect SNPs depends upon the differences in the melting temperature (Tm) of the amplified sequence of DNA (amplicon) brought about by the differences in nucleotide composition due to the presence of SNPs.

Question 23

What are some applications of PCR SNP detection?

Answer 23

• Antibiotic resistance testing - used to discriminate between 2 organisms that have resistance to an antibiotic as long as the resistance is due to a single mutation in a particular gene
• Identification of genetic markers within a genome

Question 24

How can PCR be used to identify short tandem repeats (STRs)?

Answer 24

• Labelled primers are produced that flank individual STRs.
• A PCR reaction is then performed and the products produced by PCR are then separated on a gel by gel electrophoresis which separates the products by size.
• The label is then read to identify the size of the specific STR that it was associated with.

Question 25

What are some other applications of PCR?

Answer 25

Amplifying material prior to:

• Next generation sequencing
• Isolating individual segments of DNA prior to cloning or sequencing

Manipulating and modifying DNA e.g. Introducing mutations into a sequence of DNA