Genomics

Question 1

What does Polymerase Chain Reaction (PCR) permit?

Answer 1

• production of enormous nos. of copies of specific DNA sequence (DNA amplification)
• starts with minute quantities of initial target DNA

Question 2

How many oligonucleotide primers are required for PCR and what are they designed to be?

Answer 2

• 2 are required
• designed to be complementary to flanking sequences of target DNA segment that’s to be amplified

Question 3

What do these primers do?

Answer 3

• direct repeated cycles of localised DNA replication to produce exponential increase in copies of target sequence

Question 4

Three steps in PCR cycle

Answer 4

• heat denaturation (leaving DNA single stranded)
• primer annealing to each original strand for new strand synthesis
• strand elongation/primer extension; DNA polymerase extends the 3′ end of each primer along the template strands, also deoxynucleotide triphosphates (dNTPS) are involved
• 25-35 cycles

Question 5

What happens after the cycles?

Answer 5

• DNA amplification product can be checked by UV light following gel electrophoresis and stained with a DNA stain

Question 6

Sanger sequencing

Answer 6

• PCR-like reaction but with just single oligonucleotide primer
• utilises DNA template to generate series of detectable single-stranded fragments of increasing length

Question 7

What is the chemical reaction set up in Sanger sequencing?

Answer 7

• DNA template (often a purified PCR product)
• thermostable DNA polymerase
• single oligonucleotide primer
• 4 dNTP substrates (dATP, dCTP, dGTP, dTTP)
• small quantity of dideoxynucleotide triphosphates (ddNTPs), each with different attached fluorescent label

Question 8

DNA polymerisation in Sanger sequencing

Answer 8

• incorporates dNTPs, generating new DNA strand that is exactly complementary to DNA template
• ddNTP eventually incorporated by chance instead of dNTP
• at this point, further elongation of chain is halted as ddNTP lacks 3’OH group necessary for addition of next nucleotide

Question 9

Result of DNA polymerisation in Sanger sequencing

Answer 9

• series of partially completed product chains each with particular fluorescent ddNTP at its 3’ end

Question 10

What happens after DNA polymerisation in Sanger sequencing?

Answer 10

• product molecules separated by length by gel electrophoresis
• DNA is negatively charged, so the oligonucleotides will be pulled toward the positive electrode on the opposite side of the gel
• then electronically detected and identified as they migrate through gel (by wavelength of emitted fluorescence, upon laser excitation)

Question 11

What happens after gel electrophoresis in Sanger sequencing?

Answer 11

• reading the gel to determine the sequence of the input DNA
• Because DNA polymerase only synthesizes DNA in the 5’ to 3’ direction starting at a provided primer, each terminal ddNTP will correspond to a specific nucleotide in the original sequence (e.g., the shortest fragment must terminate at the first nucleotide from the 5’ end, the second-shortest fragment must terminate at the second nucleotide from the 5’ end, etc.)
  -Therefore, by reading the gel bands from smallest to largest, we can determine the 5’ to 3’ sequence of the original DNA strand

Question 12

Limitations of Sanger sequencing

Answer 12

• Expensive, low throughput
• Labour intensive
• Low sensitivity – e.g. detection of mutations in cancer need to be present in >30% of cells