PCR and diagnostics

Question 1

What is polymerase chain reaction (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.
• In PCR a partially double stranded structure is formed by annealing single stranded DNA so in order to achieve this the double stranded template needs to be denatured first (separated) into single stranded molecule

Question 2

Define chain reaction

Answer 2

• a series of events each one depending on the preceding event to sustain itself.
• typically leads to exponential(2n) increase in the number of events occurring in a sequence

Question 3

What determines the sequence amplified (amplicon)?

Answer 3

• sequence at the end of a DNA sequence
• primer that is complementary to the end of sequence is used
• primers hybridise to these ends and form duplex.

Question 4

What is DNA polymerase and what does it do?

Answer 4

• enzyme that creates DNA molecules by assembling nucleotides.
• DNA polymerase recognises duplexes primers hybridise and forms initiation complexes around them.

Question 5

What determines the specificity of amplification?

Answer 5

1. uniqueness of the sequences at the end of amplificon, complementarity of primers to these sequences.
2. Tm used for hybridisation so high stringency helps make perfect matches and avoids mismatches (anneal temperature)

Question 6

how is amplification exponential?

Answer 6

• using 2 primers

- each complimentary to each end.

Question 7

What is annealing?

Answer 7

• annealing is the process of hybridisation by using template single strand and primers and polymerase to form a double stranded DNA molecule.

Question 8

What is renaturation?

Answer 8

when the denatured DNA re-attaches and new complementary strand can’t be formed.

Question 9

How can you eliminate the competition of annealing of primer Vs. renaturation of the template?

Answer 9

• template: low copy number
• primer : very high copy number
  => this allows the annealing to be favoured over renaturation.

Question 10

What are some basic rules of PCR?

Answer 10

=> synthesis a new nucleic acid strand by copying a DNA molecule
=> can not copy RNA nor make RNA
=> RNA must be first converted to DNA before it can be amplified by reverse transcription.

Question 11

What does the reaction require?

Answer 11

1. a template strand with a primer (20-30 bases)
2. (dATP, dGTP, dCTP, dTTP) deoxynucleotidetriphosphastes
3. Mg2+
4. a roughly neutral pH

Question 12

What is the cyclical process?

Answer 12

=> reaction depends on transition between three states reliant of hybridisation of primers and formation of partial duplex.
1. denatured (template becomes single stranded)

2. annealed ( formation of initiating template)
3. elongation by polymerase

Question 13

Why does the DNA polymerase have to be themostable?

Answer 13

• for PCR to work extreme heating is used

- polymerase from a thermophilic bacterium , thermus aquaticus is often used (Taq polymerase)

Question 14

outline the process of PCR?

Answer 14

1. Denaturation (95)
   => double stranded DNA is heated for about 15 sec , this breaks the H bonds holding them .
2. annealing (55), at Tm of primer.
   => DNA primers added
   => each primer binds to 3’ end of the DNA sequence (amplicon)
   => lower temp allows hybridisation of primer
3. elongation (72) = temp resistance DNA polymerase (Taq) + dNTPs are added .
   => DNA polymerase works in 5’ -> 3’ direction.

Question 15

What are some examples of diagnostics PCR is used in?

Answer 15

• presence /absence of TB
• differentiating between closely related organisms
• identifying ppl positive to covid.

Question 16

What is the shape of curve showing the reaction?

Answer 16

• sigmoidal curve
• reaction is inhibited as it goes on bc :
  1. reactants are used up
  2. reaction becomes too acidic.

Question 17

Why can’t end point of the reaction be used to determine the number of template copies/quantitive output?

Answer 17

• same end point as amplification becomes rate limited

- this is independent of starting conc of template.

Question 18

What can we use to determine copy number then?

Answer 18

• position of the curve
• more copies when the curve is in the left of the graph.
• we use real time PCR

Question 19

What factor does PCR use to detect SNP?

Answer 19

1. high resolution melting (HRM) : differences in melting temp (Tm) dependent on nucleotide composition.
2. probe based version of qPCR : sometimes referred to as allelic discrimination where specific binding of the probe to be amplified region containing SNP is detected.

Question 20

What are common applications of PCR?

Answer 20

• antibiotic resistance testing - TB

- identification of genetic markers - drug sensitivity/ catbolism, markers of disease (cancer)

Question 21

What is the application of PCR in forensics and law?

Answer 21

• parentage or kinship :immigration and inheritance
• identification : military casualties
• matching biological materials from two sources
• authentification of biological material

Question 22

Why can short tandem repeats (STRs) or microsatillites be used in forensics?

Answer 22

• STRs are 2- 5 or more bases in length repeated many times 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 individual genome.

Question 23

What are other applications of PCR?

Answer 23

1. amplifying material
   => before next generation sequencing
   => isolating individual segments of DNA prior to cloning or sequencing.
2. Manipulating and modifying DNA
   => introducing mutations into a sequence of DNA
   => modifying sites compatible with cloning vectors