PCR

Question 1

Polymerase Chain Reaction (PCR) Outline

Answer 1

Molecular technique to generate millions of copies of a DNA fragment template (not entire genome). DNA fragment formed by restriction enzymes

Question 2

PCR Limitation

Answer 2

Need to understand the structure of fragment before PCR, to select specific primer

Question 3

3 Stages of PCR Cycles

Answer 3

Denaturation, Annealing and Extension

Question 4

Denaturation Outline

Answer 4

Temp raised to 94-97 C. Breaks H bonds in DNA helix, forms 2 separate strands of DNA

Question 5

Annealing Outline

Answer 5

Temp is lowered to 54C. Selective primers binds

Question 6

Extension Outline

Answer 6

Temp is raised to 72C. DNA polymerase extends strands by free nucleoslides triphosphates (5’-3’)

Question 7

Considerations When Choosing DNA Polymerase for PCR

Answer 7

Must be thermoresistant must survive all temperatures of cycle Eg taqu polymerase

Question 8

Considerations When Choosing Primers for PCR

Answer 8

Must be thermoresistant, if multiple must have shared annealing temp and primers must be complementary/selective to bases on DNA. 18-24nt long, melting temps 50-60 C, should gave a strong GC clamp

Question 9

What should you minimise complementary base pairs on primers

Answer 9

Primer Dimer

Question 10

Self Dimers Outline

Answer 10

2 identical primers form a dimer, binding complementary base pairs

Question 11

Cross Dimers Outline

Answer 11

Forward and reverse primers, complementary base pairs bind

Question 12

Hairpin Loops

Answer 12

Complementary Base Pairs on same strand of primer causes it to loop around

Question 13

How is primer-dimer risk evaluated

Answer 13

Gibb’s Free Energy (delta G). Lower the Gibb’s Free Energy the more likely the structure is to form. Assumed primer is unusable if (G delta <-9 kcal/mole)

Question 14

Basic Local Alignment Search Tool (BLAST) Outline

Answer 14

Database used to map gene fragments and research compatibility of primers

Question 15

PCR Troubleshooting

Answer 15

Mg conc (low = no product, high = undesired product), higher annealing conc, prevent dimer formation (DMSO) and ensure min conc of DNA for DNA template is made

Question 16

Reverse Transcription PCR (RT-PCR) outline

Answer 16

Reverse transcriptase convert RNA to complementary DNA. Using cDNA PCR continues as normal. Used in Covid tests (fluorescent tag is integrated into cDNA), if test comes back negative there usn’t a high enough viral RNA conc to indicate infection

Question 17

Quantitative/real - time PCR (qPCR)

Answer 17

Quantifies volume of template DNA in a sample. PCR with fluorescence reporter molecule. Exponential graph; evaluate based on Cq value

Question 18

What Does Cq value indicate in qPCR

Answer 18

Point at which there’s a high enough volume of DNA + fluorescence reporter molecules, so that fluorescence exceeds background signal. Lower Cq values = lower no of PCR cycle needed = higher amount of template data already there

Question 19

Multiplex PCR

Answer 19

Multiple samples run at once. Mian consideration; primer compatibility