PCR in Diagnostics

Question 1

Describe the principles of PCR amplification of DNA fragments

Answer 1

• cyclic process: chain reaction = more events from one successful reaction
• high stringency conditions with both primers being complementary to ends of each segment with identical Tm = annealing of specific sequence at same time = prevention of mismatch
• product is template for each future successive round, due to the forward primer being extended and hybridised.
• molar XS of primer = drives annealing to be favoured over renaturation
• thermostable DNA polymerase: able to retain function after heating fluctuations

Question 2

Describe the reagents required for a PCR reaction to take place

Answer 2

DNA dependant DNA Polymerase
- detects initiation complex formed by single strand and nascent strand formed during annealing
- template of opposing primers with 3’ and 5’ overhangs
- neutral pH (prevent denaturing and optimum enzyme conditions)
- Mg2+ ions (co-factors)
- deoxynucleotide triphosphates (dATP, dGTP, dTTP)

Question 3

How is specificity of amplification ensured?

Answer 3

related to stringency and complementarity
formation of h-bonds and identical Tm of primer duplex

Question 4

Describe these modifications to the basic technique:

• Quantitative PCR (qPCR)
• SNP detection using High-resolution melting or qPCR
• Forensic use of microsatellite genetic markers (STRs) (amplifying genetic markers to detect kin)

Answer 4

Why are modifications used?
- because the yield plateaus due to rate limiting conditions which are independent of starting concentration (different conc of starting material can still have same end point)

qPCR = elongation of template = intercalation = fluorescence = quantitate molecule number at start of reaction

SNP detection: determine number of variants / single nucleotide alterations

Modification = difference in Tm (HRM)
Allelic discrimination: detection of amplified region with SNP and specific binding of probe

STR = 2-5 base pair repetitions along genome at specific locations. polymorphic nature means repeats can be used to detect inheritance/relations

Question 5

Define PCR

Answer 5

• enzyme based method
• amplify DNA fragments
• thermal acting DNA polymerase in cyclical process

Question 6

Describe some applications for PCR

Answer 6

• H1N1 human influenza types / swine flu
• Sars-COV-2 detection
• detection of TB + plan further treatment