Sequencing

Question 1

By who and how was DNA sequencing developed

Answer 1

It was developed when Sanger and his team developed some techniques for sequencing nucleic acids from viruses and then bacteria.

Question 2

What did the original sequencing process made by Sanger involve

Answer 2

Radioactively labelling of bases and gel electrophoresis on a single gel

Question 3

What was a problem with the first version of sequencing and how did Sanger complete this.

Answer 3

The process was carried out manually so it took lots of time but eventually Sanger and his team developed Sanger sequencing.

Question 4

What is a feature of DNA sequencing techniques

Answer 4

They are constantly being improved

Question 5

What is an example of an improvement that has been made to DNA sequencing

Answer 5

The swapping of radioactive tags for fluorescent tags leading to the speeding up and automation of the process.

Leading to the capillary version of Sanger sequencing

Question 6

What occurred in 1990

Answer 6

The Human genome project was established where scientists from all around the world worked to map the entire genome making the data freely available to scientists.

Question 7

What was a feature of the HGP

Answer 7

It was completed 2 years earlier and under budget due to automation and the development of higher power computers.

Question 8

What are the principles of DNA sequencing

Answer 8

The DNA is cropped into fragments ad each fragment is sequenced

It involves terminator bases which are modified versions of A,T,C,G which stops DNA synthesis when they are included.

Question 9

What is the first step in the process of DNA sequencing

Answer 9

The DNA for sequencing is mixed with a primer, DNA polymerase and an excess of normal nucleotides and terminator bases.

The mixture is placed in a thermal cycle - a piece of equipment used for PCR, that rapidly changes temperature at intervals in repeated cycles.

Question 10

In the thermal cycles what are the key temperatures and what occurs at them

Answer 10

96*C the double stranded DNA sequence are split into single strands

At 50*C the primers anneal to the DNA

At 10*C DNA polymerase is added to build up the new DNA strands by adding nucleotides with complementary bases to the single stranded-template.

Question 11

What is step 2 of DNA sequencing (1) (terminator)

Answer 11

Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is terminated as no more bases can be added

As these are added in small amounts and are random it results in fragments of many different lengths depending on where these bases have been added.

Question 12

What is step 3 of DNA sequencing (2) (capillary)

Answer 12

They are then separated by their length by capillary sequencing

The fluorescent markers are used to identify the last base in each sequence

Lasers detect the different colours and thus the order of the sequence

Question 13

What is the last step of DNA sequencing (1)

Answer 13

The order of bases in the capillary tubes show the sequence of the new complementary strand of DNA which has been made. This is used to build up the sequence of the original DNA strand.

Question 14

What is the last step of DNA sequencing (2) (computer)

Answer 14

The data is then fed into a computer reassembling the genome by comparing fragments and finding the areas of overlap between them

Once assembled they can identify parts of the genome used to code for specific characteristics.

Question 15

What has technological development lead to

Answer 15

New automated processes

Question 16

What is being used instead of gel or capillaries for DNA sequencing now

Answer 16

The sequencing reaction takes place on a plastic slide known as a flow cell

All the clusters are then sequenced and imaged at the same time so the technique is known as ‘massively parallel sequencing’