6.3.1: DNA sequencing

Question 1

In 1969, a gene was isolated from a bacterial chromosome and in 1972, a Belgian molecular biologist sequenced a gene that codes for the protein coat of a virus, MS2. Why were these processes extremely slow and only suitable for very short genes?

Answer 1

• Because both scientists worked from the mRNA transcribed from the gene, and not the raw DNA.
• RNA is unstable.

Question 2

In 1975, what did British biochemist Fred Sanger develop?

Answer 2

a method that ultimately allowed scientists to sequence whole genomes.

Question 3

Sanger’s approach was to use a single strand of DNA as a template for four experiments in separate dishes. Wha did each dish contain?

Answer 3

• The four bases A, T, C and G
• DNA polymerase
• To each dish, a modified version of one of the DNA bases was added.

Question 4

How were the DNA bases modified in the Sanger approach?

Answer 4

• In such a way that once incorporated into the synthesised complementary strand of DNA, no more bases could be added.
• Each modified base was also labelled with a radioactive isotope.

Question 5

In the Sanger method, once you have thousands of fragments of DNA bases of varying lengths, what do you do with them?

Answer 5

• The DNA fragments are passed through a gel by electrophoresis.
• Smaller fragments travel further so the fragments are sorted by length.
• The nucleotide base at the end of each fragment is read according to its radioactive label.

Question 6

Why is the Sanger method of DNA sequencing good?

Answer 6

-It’s efficient and safe.

Question 7

Why is the Sanger method of DNA sequencing not good?

Answer 7

• Time-consuming (he had to count off the bases one by one from the bands in a piece of gel)
• Costly

Question 8

What was the first DNA-based organism to have its genome sequenced?

Answer 8

-Sanger sequenced the genome of a phage virus (Phi-X174)

Question 9

What did Sanger do in 1981?

Answer 9

-He published his sequence of the human mitochondrial genome, consisting of 37 genes and 16,569 base pairs.

Question 10

What did scientists sequence in 1984?

and 1995?

Answer 10

• The 170 kilobase pair-long genome of the Epstein-Barr virus.
• The genome of the bacterium Haemophilus influenzae.

Question 11

How is the gene to be sequenced in the sanger method cloned?

Answer 11

• Using restriction enzymes from a bacterium, the gene to be sequenced was isolated.
• The DNA was then inserted into a bacterial plasmid (the vector) and then into an Escherichia Coli bacterium host that, when cultured, divided many times, enabling the plasmid within the DNA to be copied many times.
• These lengths of DNA can then be isolated and sequenced.

Question 12

What was developed at the California Institute of Technology in 1986?

Answer 12

The first automated DNA sequencing machine, based on Fred Sanger’s method.

Question 13

How is the automated SNA sequencing machine slightly different to Frend Sanger’s method?

Answer 13

• Fluorescent dyes instead of radioactivity were used to label the terminal bases.
• These dyes glowed when scanned with a laser beam and the light signature was identified by computer.
• Dispenses the need for technicians to read autoradiograms.

Question 14

The development of ____ _________ _________ has allowed us to sequence the DNA of many organisms quickly and the cost of analysing the genomes of individual humans has decreased greatly.

Answer 14

High throughput sequencing

Question 15

When was pyrosequencing developed and how is it different to the Sanger method?

Answer 15

• 1996

- Uses sequencing by synthesis, not by chain termination as in the sanger method.

Question 16

Briefly, what does pyrosequencing involve?

Answer 16

-A single strand of DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting, by light emission, which base was added at each step.

Question 17

There are 8 steps in pyrosequencing.

Step 1:

Answer 17

1. A long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser.

Question 18

There are 8 steps in pyrosequencing.
Step 1: A long length of DNA to be sequenced is mechanically cut into fragments of 300-800 base pairs, using a nebuliser.
Step 2:

Answer 18

2. These lengths are then degraded into single-stranded DNA (ssDNA). These are the template DNAs and they are immobilised.

Question 19

There are 8 steps in pyrosequencing.
Step 2: These lengths are then degraded into single-stranded DNA (ssDNA). These are the template DNAs and they are immobilised.
Step 3:

Answer 19

3. A sequencing primer is added and the DNA is then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’phosphosulfate (APS) and luciferin.
   - Only one of the four possible activated nucleotides, ATP, TTP, CTP, GTP is added at any one time and any light generated is detected.

Question 20

There are 8 steps in pyrosequencing.
Step 3: A sequencing primer is added and the DNA is then incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, apyrase and the substrates adenosine 5’phosphosulfate (APS) and luciferin.
-Only one of the four possible activated nucleotides, ATP, TTP, CTP, GTP is added at any one time and any light generated is detected.
Step 4:

Answer 20

4. One activated nucleotide (a nucleotide with two extra phosphoryl groups), such as TTP (thymine triphosphate), is incorporated into a complementary strand of DNA using the strand to be sequenced as a template.

Question 21

There are 8 steps in pyrosequencing.
Step 4: One activated nucleotide (a nucleotide with two extra phosphoryl groups), such as TTP (thymine triphosphate), is incorporated into a complementary strand of DNA using the strand to be sequenced as a template.
Step 5:

Answer 21

5. As this happens, the two extra phosphoryls are released as pyrophosphate (PPi).

Question 22

There are 8 steps in pyrosequencing.
Step 5: As this happens, the two extra phosphoryls are released as pyrophosphate (PPi).
Step 6:

Answer 22

6. In the presence of APS, the enzyme ATP sulfurylase converts the pyrophosphate into ATP.

Question 23

There are 8 steps in pyrosequencing.
Step 6: In the presence of APS, the enzyme ATP sulfurylase converts the pyrophosphate into ATP.
Step 7:

Answer 23

7. In the presence of this ATP, the enzyme luciferase converts luciferin to oxyluciferin.

Question 24

There are 8 steps in pyrosequencing.
Step 7: 7. In the presence of this ATP, the enzyme luciferase converts luciferin to oxyluciferin.
Step 8:

Answer 24

8. This conversion generates visible light which can be detected. by a camera.
   - The amount of light generated is proportional to the amount of ATP available and, therefore, indicates how many of the same type of activated nucleotide were incorporated adjacently into the complimentary DNA strand.

Question 25

What happens to leftover activated nucleotides in pyrosequencing?

Answer 25

Unincorporated activated nucleotides are degraded by apyrase and the reaction starts again with another nucleotide.

Question 26

One million reads occur simultaneously, so a 10-hour run generates 400 million bases of sequencing information. What assembles these sequences into longer sequences.

Answer 26

Software packages.

Question 27

Why has a branch of biology called bioinformatics been developed?

Answer 27

• To store the huge amounts of data generated from this research.
• It would have been impossible to store and analyse these data prior to computers and microchips.
• Software packages are specially designed for this purpose.