Wright 5 - DNA Sequencing and PCR

Question 1

What are the minimum requirements for DNA synthesis in vitro?

Answer 1

• A ssDNA template
• A DNA primer annealed to the template
• DNA polymerase
• Deoxyribonucleoside triphosphates

Question 2

What are two strategies for DNA sequencing? Which one is used today?

Answer 2

• Chemical cleavage method
• Enzymatic dideoxy chain terminating method

Enzymatic dideoxy chain terminating method is used today, it is also semi automated

Question 3

What type of bond does DNA polymerase make and how?

Answer 3

A phosphodiester bond, by a nucleophilic attack of the 3’ hydroxyl group on the alpha phosphorous atom of the incoming dNTP

Question 4

What is the difference between a deoxyribonucleoside triphosphate (dNTP) and a dideoxyribonucleoside triphosphate (ddNTP)?

Answer 4

A dNTP extends a DNA strand during DNA synthesis. A ddNTP terminates synthesis. A dNTP has a 3’ hydroxyl group and a ddNTP does not.

Using ddNTPs, daughter strands of different lengths can be produced.

Question 5

What are the steps of the dideoxy chain terminating method?

Answer 5

1. ssDNA of unknown sequence serves as a template
2. A radioactively labeled primer along with DNA polymerase and ddNTPs are added. Only one ddNTP (eg. dATP or dCTP etc.) is used per reaction mixture (4 reaction mixtures in total, one for each dideoxyribonucleoside triphosphate)
3. Gel electrophoresis is done on the four reaction mixtures (all four on same gel)
4. Longer fragments contain more nucleotides than shorter.

Long fragment end is 3’ for new stand, short fragment end is 5’ for new strand. The sequence is read from the bottom of the gel to the top (wells) in 5’ to 3’ direction. The new strand is then used to deduce sequence of the template strand.

Question 6

Who developed the polymerase chain reaction (PCR)?

Answer 6

Dr. Kary Mullis

Question 7

True or false, both DNA sequencing and PCR require DNA synthesis.

Answer 7

True

Question 8

What are the 6 steps of PCR?

Answer 8

1. Denaturing to ssDNA and annealing of primer
2. Extension of primer via Taq DNA polymerase
3. Repeat cycles to amplify a specific sequence of DNA

Question 9

Why is Taq polymerase from Thermus aquaticus used for PCR?

Answer 9

Because it does not denature at high temperatures

Question 10

Amplification of a targeted DNA molecule by PCR is in what type of graph? (eg. exponential, logarithmic etc.)

Answer 10

There is a linear phase, then an exponential phase and then a plateau, so all in all it makes a sigmoidal shape

Question 11

What are four advantages of PCR over older technologies?

Answer 11

• Rapid cloning of DNA fragments (need to know the sequence of part of the DNA for PCR)
• Rapid detection of genetic disease and DNA typing (or fingerprinting)
• Minute amount of DNA required for PCR
• Analysis of ancient DNA for historical and archaeological studies