21.2 DNA Sequencing and Analysis

Question 1

What is DNA sequencing?

Answer 1

The process of determining the sequence of nucleotides in a piece of DNA.

Question 2

What did the Sanger sequence involve?

Answer 2

Radioactive labeling of bases and gel electrophoresis on a single gel.

Question 3

How was the Sanger sequence method refined?

Answer 3

• Swapping of radioactive labels with fluorescent tags.
• This allowed for the automation of the sequencing method.
• Lead to capillary sequencing method (used in the human genome project).

Question 4

What are the ingredients needed in DNA sequencing?

Answer 4

• Terminator bases
• DNA polymerase
• Primers
• DNA nucleotides.
• Template DNA strand.

Question 5

What happens in the first step of the DNA sequencing method?

Answer 5

DNA to be sequenced is mixed with primers, nucleotides and terminator bases

Question 6

What happens in the second step of the DNA sequencing method?

Answer 6

Mixture is placed in a thermal cycler (used in PCR) and heated to 96°C to separate template strands, cooled to 50°C to allow primers to anneal to each DNA template strand, and heated to 60°C to allow DNA polymerase to extend from primers via complementary base pairing on each template strand.

Question 7

What happens in the third step of the DNA sequencing method?

Answer 7

A terminator base (modified nucleotide) is added each time to stop the synthesis of DNA so no more bases can be added. The cycle repeats, producing DNA fragments of all possible lengths.

Question 8

What happens in the fourth step of the DNA sequencing method?

Answer 8

DNA fragments are separated according to lengths by capillary sequencing (like electrophoresis). Terminator bases contain a fluorescent marker which lasers detect with UV light.

Question 9

Why are there fluorescent tags on each terminator base?

Answer 9

They are used to identify the final base on each fragment and allow the sequences to be determined.

Question 10

What happens in the fifth step of the DNA sequencing method?

Answer 10

The data from the sequencing is fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them.

Question 11

What are the uses and limitations of DNA sequencing?

Answer 11

• Used in sequencing pieces of DNA such as plasmids or DNA copied in PCR.
• Method is expensive and inefficient for larger projects.
• New larger scale sequencing techniques are faster and cheaper.

Question 12

What is next generation sequencing?

Answer 12

A method for analysing genetic material that can quickly sequence large amounts of DNA or RNA. Quicker, more efficient and cheaper than Sanger sequencing.

Question 13

Describe next generation sequencing.

Answer 13

• Sequencing takes place on a flow cell (plastic slide) rather than gel or capillaries.
• DNA fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments.
• Still uses coloured fluorescent terminator bases.