6.3.1(DNA sequencing)

State the name of the method used for DNA sequencing

• Chain termination method - Sanger method

Describe the requirements for the sanger method

• Single stranded DNA template

• DNA primer

• DNA polymerase

• normal nucelotides

• Modified dideoxy nucleotides - terminator genes

Describe how terminator genes prevent DNA strand elongation

• They are modified so the 3’ carbon on the deoxyribose contains no -OH group

• This means that the nucleotides are not able to form phosphodiester bonds and therefore the DNA strand is terminated

Describe the process of DNA sequencing using Sanger’s method

• A single template DNA strand is formed by heating up the DNA to 95C to break the hydrogen bonds between complementary bases

• A primer is then attached

• There are 4 tubes with contain ddNTP - modified nucleotides - with each tube having its own modified base. e.g.. one tube with A, One tube with T, etc,

• Add the template strand to the solutions. This will make many strands of different lengths. This is because the ddNTPs are added at random intervals

• The many random strands are separated from the template by heating. These strands vary in length

• They are then put through gel electrophoresis. The shortest strands will travel the furthest, and a code can be read off them

• ddNTPs are radioactively labelled and so are then identified

Describe how DNA sequences were read before autoradiograms

• Fluorescent dyes instead of radioactivity were used to label terminal bases.

• The dyes would glow when scanned with a laser beam and the light signature was identified by computer

Describe the downsides of using the sanger method for DNA sequencing

• Uses a human to count off the bases one by one:

  • Time consuming

  • Costly

Describe how the advancement of technology has lead to improved DNA sequencing methods

• Technological advancements have lead to the development of an autoradiograph, which is used to identify how far DNA bands have travelled.

  • Allowed for the development of Pyrosequencing - high throughput sequencing
    

Describe pyrosequencing

• It is an automated technology

• Involves synthesising 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 to each step

• Thousands to millions of DNA molecules can be sequenced at the same time - in parallel

• Software packages assemble these sequences into longer sequences

Describe the benefits of using pyrosequencing over using Sanger’s method

• Thousands to millions of DNA molecules can be sequenced at the same time - in parallel

• Methods can be 1000x faster than older methods

• Costs are reduced to reduction of time needed for sequencing