Lecture 4

Question 1

What knowledge can we get from DNA sequencing?

Answer 1

• Understanding of how genes and proteins work
• Understanding of how different gene products influence the activity of each other within the context of a whole organism

Question 2

What are some of the ways that the knowledge taken from DNA sequencing is used?

Answer 2

• Used to establish phylogenetic relationships between organisms
• Used to look for and study genetic disease mechanisms (cancer and other inherited diseases)
• Used in the world of forensics

Question 3

What happens during the process of DNA sequencing?

Answer 3

The process involves determining the unknown bases of the DNA strand and also the sequence in which they occur

Question 4

What are the 5 major stages of DNA sequencing (Sanger sequencing/ chain termination method)?

Answer 4

1. Preparation of the reaction mixtures
2. DNA synthesis
3. Gel electrophoresis
4. Autoradiography
5. Determination of the new strand sequence and deduction of the template sequence

Question 5

What goes into step 1 (preparation of the reaction mixtures) CTM?

Answer 5

4 separate reaction mixtures are set up, all including the following:

• Single stranded DNA with the UNKNOWN sequence that we are trying to determine (within this there needs to be the known sequence as well for the primer to bind to)
• Radioactively labelled primer (a short sequence of single stranded DNA that compliments and will bind to our known sequence of DNA)
• DNA polymerase (to synthesise the sequence)
• Normal nucleotides (dNTP’s) [the nucleotides that will be added to the primer in order to build up the new DNA strand]

Question 6

What is the difference between a deoxynucleotide (normal nucleotide) and a dideoxynucleotide?

Answer 6

On the 3’ carbon of the molecule the deoxynucleotide possesses an OH group where as the dideoxynucleotide doesn’t have one at all

Question 7

Why is the OH group on the 3’ of the deoxynucleotide important?

Answer 7

It allows for continual synthesis as the 5’ of another dNTP would bind to this location

Question 8

Why does the H group on the 3’ of the dideoxynucleotide cause a halt in the synthesis of a new DNA strand?

Answer 8

It is a modified nucleotide and doesn’t possess the OOH group that another dNTP would use to bind and extend the DNA strand. The H group does allow the necessary reactions to take place to allow another nucleotide to bind

Question 9

Why are there 4 separate tubes that are set up to carry out the chain termination method?

Answer 9

Because ddNTPs are used to terminate the chain of DNA to allow us to know the DNA sequence, one of the ddNTPs are added per tube. Either ddATP, ddCTP, ddGTP or ddTTP

Question 10

What happens during step 2 (DNA synthesis) CTM?

Answer 10

• The primer that is radioactively labelled binds to the short known sequence on the single stranded DNA with the unknown sequence
• DNA polymerase can then begin to synthesise (5’ to 3’) the DNA, without the primer this wouldn’t be possible

Question 11

How does the DNA polymerase catalyse the adding of new bases?

Answer 11

For example a G on the template strand needs a C

• DNA polymerase catalysed a dCTP and releases 2 phosphate groups allowing the base to bind to the OH group of the 3’ of the nucleotide

Question 12

What happens if a ddNTP is used during synthesis rather than a dNTP?

Answer 12

The sequence will be terminated, the ddNTPs don’t possess the OH group on the 3’ of the molecule and therefore no other nucleotides can bind onto it

Question 13

What happens during step 3 (gel electrophoresis) CTM?

Answer 13

The lengths of DNA are separated through size and charge, the bigger lengths of DNA being found in bands towards the top of the gel and smaller towards the bottom, this shows for each of the 4 tubes where the ddNTP was in the sequence

Question 14

What happens during step 4 (autoradiography) CTM?

Answer 14

The fragments on the gel cannot be visualised directly, an X-ray film must be placed on top of the gel to allow this to happen this is why the ddNTPs are labelled so we can see where they are when using an X-ray film

Question 15

What happens during step 5 (Determination of the new strand and deduction of the template sequence) CTM?

Answer 15

The gel electrophoresis is read which allows us to visualise what bases are at what positions in the DNA sequence, however this is the new strand so working with this new strand we can apply complementary base pairing and work out the original unknown template strand

Question 16

What is automated capillary sequencing?

Answer 16

Same method as the chain termination sequence with the ddNTPs however the ddNTPs are all fluorescently labelled different colours so that all of the ddNTPs can be run in the same tube instead of 4 different ones. Instead of using gel electrophoresis capillary electrophoresis is used, a long thin tube with a laser through it which picks up the colours of the differently labelled fluorescent ddNTPs which then gets outputted by a computer program as a series of coloured peaks

Question 17

What are the advantages of using automated capillary sequencing over the chain termination method?

Answer 17

• More time and cost effective
• Less pipetting steps which means less room for human error (also automated)
• Do not need to label the primers because already for ddNTPs labelled
• More accurate and more reliable

Question 18

What are some of the technologies that are put under the umbrella term ‘Next Generation Sequencing’?

Answer 18

• Roche 454 pyrosequencing - DNA template strand is attached to a microscopic bead, one nucleotide at a time is added into the reaction, the nucleotide is bound, when it bind it releases a pyrophosphate then enzymes use ATP to produce a light source. The amount of light is quantifiable and tells you which nucleotide has bound
• Ion torrent
• PacBio
• Illuminia Miseq/Hiseq

Question 19

What are the advantages of using illumina sequencing?

Answer 19

• Can detect any base call errors (incorrect nucleotides incorporated into the strand)
• Sensitive
• Specific
• Low cost