Manipulating Genomes

Question 1

What is DNA sequencing used for?

Answer 1

DNA sequencing allows for nucleotide base sequences of an organisms genetic material to be identified and recorded

Question 2

What is a dideoxynucleotide

Answer 2

A nucleotide which OH group has been deoxygenated to just hydrogen meaning it can not form phosphodiester bonds with other nucleotides

Question 3

What is sangers method of DNA sequencing?

Answer 3

• A chain termination method of DNA sequencing which uses modified nucleotides known as dideoxynucleotides
• Dideoxynucleotides can pair with nucleotides using a primer at the start of the template strand during the DNA replication
• These dideoxynucleotides pair with a nucleotide that have a complimentary base
• When DNA polymerase encounters a didieoxynucleotide on the developing strand it stops replicating.

Question 4

Process of Sangers sequencing method

Answer 4

1. Firstly many copies of a single stranded piece of DNA are used as templates to sequence
2. Next a primer is added so that the enzyme DNA polymerase can bind and start copying the DNA strand
3. The DNA and primers are added to 4 identical test tubes with free nucleotides
4. Dideoxynucleotides are also added and randomly incorporated into the growing chain.
5. This stops Polymerase from forming phosphodiester bonds
6. In each tube many copies of the DNA molecule are made but at different lengths.
7. The 4 tubes are then loaded into a gel and seperated by gel electropheresis.
8. The banding pattern can then be read to work out the order of bases in the DNA sequence

Question 5

What is Automated dideoxy sequencing?

Answer 5

• Still uses chain termination with dideoxynucleotides
• Each different nucleotide is labelled with a different colour fluorescent
• There is no need for 4 different parallel gels, all are loaded into one capillary tube
• A laser reads the colour each dideoxynucleotide as it exits the capillary tube
• The readout shows emissions of coloured light peaks showing the order of bases on a chromatograph

Question 6

What is pyrosequencing?

Answer 6

Using a flash of light released every time a base is added to work out the base sequence.
Process:
1. A long base of a dna sequence is mechanically cut by a nebuliser
2. these length are then degraded to ss dna
3. These are template DNAs and are immobilised
4. A sequenciing primer is added and the DNA is incubated with enzymes
5. Only one of four possible activated nucleotides is added at a time
6. Any light generated is detected
7. Two extra phosphates are released as pyrophosphate
8. The enzyme ATP sulfurylase then converts Pyrophosphate into ATP
9. In the presence of ATP the enzyme luciferase converts luciferin into oxyluciferin
10. Creates visible light to be detected by a camera, volume of light created is proportional to the amount of ATP available which indicates which nucleotide base is there