PCR and Diagnostics

Question 1

What is the definition of PCR?

Answer 1

→ An enzyme based method to specifically amplify segments of DNA using thermal DNA polymerase in a cyclical process

Question 2

What type of increase is there in PCR?

Answer 2

→ Exponential

Question 3

What does the specificity stem from in PCR?

Answer 3

→The complementarity of the primers

Question 4

Under what conditions is PCR specific?

Answer 4

→ If annealing is undertaken at the melting point of the primers

Question 5

What does high stringency mean?

Answer 5

→ High temperature

Question 6

If you want to amplify a segment bound by a known sequence how do you do this?

Answer 6

→ Primers that are complementary to these ends

Question 7

What does DNA polymerase recognize?

Answer 7

→ A specific structure consisting of a partially double stranded DNA forming an initiation complex

Question 8

How is a partially double stranded structure formed?

Answer 8

→ By annealing a short single stranded DNA molecule to a denatured single stranded molecule

Question 9

How do you prevent annealing and renaturation happening at the same time?

Answer 9

→ Driven by having an excess of the primer

Question 10

What kind of polymerase is used in PCR?

Answer 10

→ DNA dependent DNA polymerase

Question 11

How does DNA dependent DNA polymerase work?

Answer 11

→ Synthesizes a new nucleic acid by copying a DNA molecule

Question 12

How can RNA be amplified?

Answer 12

→ It must be copied into DNA

→ By reverse transcriptase

Question 13

What are the 4 requirements of DNA polymerase to work?

Answer 13

→ Template strand with a primer
→ Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
→ Mg2+ ions
→ Roughly neutral pH

Question 14

What are the three states that PCR depends on?

Answer 14

→ Denaturation
→ Annealing
→ Native state at the optimal extension temperature and pH for enzyme activity

Question 15

What does PCR need to work?

Answer 15

→ Multiple rounds of extreme heating and cooling

Question 16

What property must the DNA polymerase have for PCR to work?

Answer 16

→ Thermostable

Question 17

What does thermostability mean?

Answer 17

→ Ability to retain activity upon repeated heating to temperatures that would destroy most enzymes

Question 18

What species of polymerase is used in PCR?

Answer 18

→ Thermophilus Aquaticus

Question 19

What are the 6 steps of PCR?

Answer 19

1) All reactants : template, primers and enzymes are mixed
2) Denaturation at 95 degrees
3) The mixture is cooled to the primer Tm and they bind to the template strand
4) The temperature is then changed to 72 degrees, a temperature which is optimal for the enzyme to work
5) An initiation complex is formed which elongates from the 3’ end which creates a second strand
6) This is repeated multiple times

Question 20

What are the kinetics of the reaction dependent on?

Answer 20

→ Depletion of reactants

→ Acidification of reaction

Question 21

Why is there acidification?

Answer 21

→ H+ ions get produced as dNTPs are added

→ Pyrophosphate is produced

Question 22

What are 3 ways PCR used in diagnostics?

Answer 22

→ Presence or absence calling TB - detection in sputum
→ Differentiating between closely related organisms ( swine flu vs human influenza)
→ Treatment starting (HIV viral load)

Question 23

What is the main limitation of PCR?

Answer 23

→ The end point is the same irrespective of the starting concentration
→ Limited by the amount of DNA polymerase

Question 24

How does real time PCR work?

Answer 24

→ Fluorescent detection of the amplification

Question 25

What is real time PCR for?

Answer 25

→ Quantifying the amount of a target DNA molecule in the sample

Question 26

What are SNPs?

Answer 26

→variations in the DNA sequence at particular locations, called single nucleotide polymorphisms
→ Difference of a single nucleotide

Question 27

What does SNP detection depend on?

Answer 27

→ Differences in melting temperature (Tm)

→ conferred due to the nucleotide composition

Question 28

What is SNP detection used for?

Answer 28

→ Antibiotic testing - TB and other organisms

→ Identification of genetic markers - drug sensitivity

Question 29

What is high resolution melting?

Answer 29

→ Tm of the amplified product is used to determine which sequence is present

Question 30

What is the probe based version of qPCR?

Answer 30

→ Specific binding of the probe to the amplified region containing the SNP is detected

Question 31

What are the 4 uses of PCR in forensics?

Answer 31

→ Parentage or kinship - immigration and inheritance
→ Identification - military casualties, missing persons or environmental disasters
→ Matching two sources - crime scenes
→ Authentication of biological material - cell lines, purity of food

Question 32

What are STRs?

Answer 32

→ STRs are 2-5 or more bases repeated at specific locations in the genome

Question 33

What do STRs provide?

Answer 33

→ A pattern of uniquely sized products

→ Molecular barcode

Question 34

What does the UK DNA database consist of?

Answer 34

→ 10 STRs

Question 35

What does forensic identification use?

Answer 35

→ STRs

Question 36

How is PCR designed so it will span many STRs?

Answer 36

→ Multiple sets of labelled primers

Question 37

What is another application of PCR in research?

Answer 37

→ Manipulating and modifying DNA

→ Introducing mutations into a sequence