PCR

Question 1

What is PCR?

Answer 1

• An enzymatic assay which allows for the amplification of specific genes from a DNA sample
• one of the key mapping techniques used in the Human Genome Project

Question 2

Explain the PCR process

Answer 2

• utilises short DNA sequences aka oligonucleotide primers, sequence = complimentary to target region of genes
  -1.DNA sample is denatured to produce single stranded DNA aka, template DNA
• Oligonucleotide primers can bind (primer annealing)
• DNA polymerase then adds nucleotide bases to the end of each primer, using the template DNA as a guide to extend the primer, therby producing new double stranded DNA
• This process= repeated for a number of cycles to amplify the desired genes targeted by the oligonucleotide primers

Question 4

What is the exponential amplification of genes?

Answer 4

• Each PCR cycle creates 2 new double stranded DNAs from each DNA molecule present
• The amount of DNA doubles with every cycle of PCR
• After 2 cycles, the conc of DNA increases by 2^2 fold
• After N cycles, PCR generates a 2N fold increase in the target DNA

Question 5

What is the DNA template?

Answer 5

• the sample DNA that contains the sequence

Question 6

What is Taq DNA polymerase?

Answer 6

• enzyme that synthesises new strands of DNA complementary to the target sequence

Question 7

What are primers?

Answer 7

• short pieces of single stranded DNA that are complementary to the target sequence
• the polymerase begins synthesising new DNA from the end of the primer

Question 8

What are nucleotides (dNTPs/ deoxynucleotide triphosphates)

Answer 8

• single units of the bases A,T ,G and C = building blocks for new DNA strands

Question 9

What is the role of MgCl2 in PCR?

Answer 9

• cofactor for activity of DNA polymerases

Question 10

What is a buffer’s role in PCR?

Answer 10

• provides a suitable chemical environment for activity of DNA polymerase
• • sterile, nuclease free water

Question 11

What is PCR reaction?

Answer 11

• performed in a thermalcycler and involves an initial DNA denaturation, followed by a number of cycles of denaturation, primer annelaing and product extension
• A final DNA extension step completes the reaction

Question 12

How to visualise PCR results?

Answer 12

• After thermal cycling, tubes are taken out of the PCR machine
• contents of tubes are loaded onto an agarose gel
• DNA is separated by size using an electirc field
• DNA is then stained
• PCR products are visible as differnet bands

Question 13

What are the advantages of PCR?

Answer 13

• Highly sensitive technique
• potential to produce millions to billions of copies of a specific product for sequencing, cloning and analysis
• can be used to test for anti-microbial resistance
• can be quickly performed in 4-8 hours
• can detect less common organisms such as viruses
• quantitative polymerase chain reaction and is used in COVID-19 testing
• method also adds fluorescent dyes to the PCR process to measure the amount of genetic material in a sample

Question 14

What are the limitations of PCR?

Answer 14

• Highly sensitive technique
• any contamination of the sample by small amounts of DNA can produce misleading results
• Primers used for PCR can anneal non-specifically to sequences that are similar but not identical to target DNA
• Incorrect nucelotides can be incorporated into the PCR sequence by the DNA polymerase