Wright Lecture 5: DNA Sequencing and PCR

Question 1

Essential biochemical features of DNA synthesis by DNA polymerase

Answer 1

1. Short segment of ds nucleic acid to serve as primer

2. 5’ to 3’

Question 2

Minimal requirement for DNA synthesis in vitro

Answer 2

1. ssDNA template
2. DNA primer
3. DNA polymerase
4. Deoxyribonucleoside triphosphates

Question 3

Chemical cleavage method

Answer 3

Strategy for DNA sequencing
Maxam and Gilbert, published in 1977
Not common now

Question 4

Enzymatic dideoxy chain termination method

Answer 4

Fred Sanger, 1975

Very popular and now semi-automated

Question 5

Fred Sanger

Answer 5

1918-2013
Welcome Trust Sanger Institute, UK
Nobel Laureat in chemistry for inventing process of protein (1958) and DNA (1980) sequencing
Published dideoxy-chain termination DNA sequence method in 1975

Question 6

Phosphodiester bond

Answer 6

Catalyzed by DNA polymerase
Nucleophile attach of 3’ OH on the innermost alpha phosphorous atom of the incoming dNTP
P-P is released

Question 7

Dideoxyribonucleoside triphosphate

Answer 7

Terminates DNA synthesis
Does not have 3’ OH for nucleophilic attach
Can be used to form daughter strands of different lengths

Question 8

Sequencing using dideoxy chain termination method

Answer 8

1. Single stranded DNA of unknown sequence as template
2. Radioactively labelled primer
3. Mix ssDNA in 4 portions with ddATP, ddCTP, ddTTP, ddGTP and DNA polymerase
4. Gel electrophoresis
5. Autoradiography to detect radioactive bands
   + end is smaller DNA sizes, read + to -
   Sequence read is complementary to template DNA strand

Question 9

Automated dideoxy sequence

Answer 9

1. Single stranded DNA fragment to be sequences added with 4 ddNTPs (each tagged with different fluorescent dye) to carry out Sanger sequencing reactions
2. Products are denatured and the DNA fragments are loaded into single well on electrophoresis gel which migrate by size
3. Fluorescent dye on DNA is detected by laser beam
4. Each fragment appears as peak on computer print out, colour shows which base it is)
5. Sequencing information is read directly into the computer which converts it into the complementary target sequence

Question 10

Why do we genetic sequence?

Answer 10

Genetic relationship of species
Genome evolution
Medical genetics

Question 11

Polymerase chain reaction (PCR)

Answer 11

Dr. Kary Mullins

Amplification of targeted DNA fragments

Question 12

Kary Mullins

Answer 12

Born 1944

Winner of Nobel Prize for Medicine and Physiology and the Japanese Prize

Question 13

PCR procedure

Answer 13

1. Denature DNA and anneal primers
2. Extend the primers with Taq DNA polymerase
3. Repeat denaturation and annealing of primers, and so on

Question 14

PCR requirements

Answer 14

ssDNA template, DNA primer, DNA polymerase, deoxyribonucleoside triphosphates
Efficiency: automate PCR with computer driven thermal cycler, Taq polymerase

Question 15

Taq polymerase (PCR)

Answer 15

From bacterium Thermus aquatics

Does not denature at high temperatures used for DNA denaturation

Question 16

Advantages of PCR

Answer 16

Rapid cloning of DNA fragments
Rapid detection of genetic disease and DNA typing
Minute amount of DNA requires for PCR
Analysis of ancient DNA for historical and archaeological genetic studies