Lecture 10: DNA Sequencing and PCR

Question 1

What does DNA sequencing do?

Answer 1

It determines the order of nucleotides/ bases in DNA.

Question 2

Who got a joint noble prize for their work in DNA sequencing?

Answer 2

Walter GIlbert and Frederick Sanger

Question 3

What is a dideoxynucleotide (ddNTP) ?

Answer 3

A modified nucleotide that prevents DNA from elongating further by terminating the chain at a specific nucleotide

Question 4

What does Sanger DNA sequencing rely on?

Answer 4

the incorporation of dideoxynucleotides into newly replicated DNA

Question 5

Name a dideoxynucleotide (ddNTP)

Answer 5

2’,3’-Dideoxyribonucleoside triphosphate, NTP

Question 6

Why are dideoxynucleotides terminators?

Answer 6

ddNTPs have no 3’-OH group. When incorporated into DNA, no more nucleotides can be added:

Question 7

Describe the principle of Sanger’s dideoxy sequencing method

Answer 7

Add:
- a single stranded template DNA
- a primer complementary to part of this template
- DNA polymerase
- a pool of normal deoxynucleotides (dATP, dTTP, DGTP and dCTP)
- a small proportion of radioactively-labelled ddATP
- Some newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA.
- The result is a nested set of new DNA molecules each of which ends in a ddA. (base is adenine)

Question 8

Describe Sanger’s sequencing method in practice

Answer 8

• Take the template (lower strand), primer, DNA polymerase, and the dNTP’s and divide into 4 aloquots.
• Add the appropriate ddNTP to each test tube. ddG to one, ddA to one, ddT to one, ddC to one.
• If you put the nested series next to each other you can separate them on the basis of size using electropheresis.
• ④Autoradiograph and read sequence from the bottom upwards.

Question 9

Describe Automated dideoxy sequencing

Answer 9

• ① template + primer + DNA Pol + all dNTPs + all fluorescent ddNTPs in ONE tube
• ②The dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that terminated the synthesis of that particular fragment.
• ③ Pass nested products through an electrophoretic system and read with lasers

Question 10

What does PCR do?

Answer 10

It amplifies a specific DNA sequence

Question 11

Who invented PCR?

Answer 11

ary Mullis in 1983, resulting in his Nobel Prize in Chemistry.

Question 12

When did Watson and Crick come up with their double-stranded DNA model?

Answer 12

In 1953

Question 13

When did Arthur Kornberg discover the first DNA polymerase?

Answer 13

In 1957

Question 14

When did Har Gobind Khorana started deciphering the genetic code?

Answer 14

In the 1960s

Question 15

When did homas Brock isolated a bacterium, Thermus aquaticus, from a hot spring in Yellowstone National Park. ?

Answer 15

In 1969

Question 16

WHen did Khorana’s group suggested that a template-primer-polymerase system could be used for copying DNA … and using two primers should lead to replication of a specific fragment of DNA (but did not show results). ?

Answer 16

In 1971

Question 17

When was Taq polymerase was isolated from T. aquaticus.?

Answer 17

in 1976

Question 18

When did Sanger get his nobel prize for ddDNA sequencing?

Answer 18

In 1977

Question 19

What is a useful feature of Taq polymerase?

Answer 19

It is thermally stable and thrives at ~ 72 degrees celcius.

Question 20

Describe the process of PCR

Answer 20

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Question 21

Is PCR an exponential amplification?

Answer 21

Yes

Question 22

Describe the process of electorpheresis

Answer 22

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Question 23

Is it true that PCR has both specificity and sensitivity?

Answer 23

Yes

Question 24

How does PCR have specificity?

Answer 24

This is provided by the primers. They are complementary to opposite strands with their 3’ ends pointing towards each other.

Question 25

How does PCR have sensitivity?

Answer 25

one target molecule can be amplified to > 109 molecules in just a few hours, because a product formed in one cycle becomes a template for the next.

Question 26

what are the applications of PCR?

Answer 26

• Making specific mutations
• Sequencing of archaic hominin genomes
• Forensic science

Question 27

Who found out that VNTRs can be used to identify people?

Answer 27

Sir Alec Jeffreys in 1984

Question 28

Describe a VNTR

Answer 28

A Variable Number Tandem Repeat (or VNTR) is a run of short repeated nucleotide sequence (each typically 3-5 bp). These can be found on many chromosomes, and often show variations in length between individuals. The number of repeats is highly variable in the population and hence individuals will usually inherit a different variant of each STR locus from their mother and father.

Question 29

Is it true that an STR can be used for personal and parental identification ?

Answer 29

Yes

Question 30

Why isn’t just a single STR useful?

Answer 30

Because a single STR may be shared by unrelated people.

Question 31

What is CODIS ?

Answer 31

a multiplex PCR is performed (multiple reactions in one tube) using specific primers for 13 different STRs on different chromosomes.