Week 4: Sanger DNA Sequencing

The Gilbert-Maxam method

• This was the first widely adopted method (from 1977)

• based on chemical modification and clevage of DNA

When was Sanger DNA sequencing the dominant method?

from the 1980s to around 2010

Is Sanger Sequencing still used today?

• Yes

• easy to set up

• accurate for short regions (up to ~500 bases)

What is the principle of Sanger Sequencing

replication of a DNA sequence in vitro using a DNA polymerase, dNTPs and dideoxy chain terminators (ddNTPs).

What are dideoxy chain terminators?

• identical to normal dNTPs except that the -OH is missing from the 3’ position of the deoxyribose part

• 3’ -OH is necessary for the subsequent addition of dNTPs to a growing DNA molecule

• if a dideoxy chain terminator is incorporated into a growing DNA molecule, that molecule will grow no more.

How is ds DNA denatured → ss?

• heating

• strong alkali e.g NaOH, formamide

What kind of gel is used for Sanger Sequencing?

• acrylamide gel

• high resolution (ss DNA by 1 bp)

What does manual Sanger sequencing use?

large flat acrylamide gels, radioactivity, and x-ray film to separate and detect reaction products

What does automated Sanger sequencing use?

• Typically uses liquid capillary gel machines and fluorescent tags to separate and detect reaction products

How does manual Sanger sequencing work?

1. Anneal oligonucleotide primer to previously denatured template strand

2. each tube has everything for polymerase to work and each tube has a different ddNTP

3. gel electrophoresis

4. count bands

Why is manual Sanger sequencing a bit rubbish?

1. difficult to pour gels without getting bubbles in

2. handling liquid acrylamide - neurotoxin

3. reactions were radioactive - radioactivity in DNA-mutagens - get absorbed into skin and destroy DNA molecules

4. error prone when reading

Other Considerations for manual sanger sequencing

• Preparation of a DNA sample for sequencing. Millions of copies of the template DNA are required – It’s normal to use PCR to generate template DNA, or clone the unknown sequence into a plasmid (see later lectures)

• Requirement for a primer to initiate the reaction. The primer is actually essential for the technique because: – the polymerase will only extend a pre-existing chain – it ensures that all the chain elongation reactions will start at the same point

• The polymerase - desirable properties including fidelity, progressiveness, thermostability. (See also notes on polymerases)

How is sequencing primers for sanger sequencing different to primers for PCR?

• Only 1 is required

• They are usually a bit shorter (~16 to 22 bases long)

Progressiveness

tendency to hold on to the DNA molecule and make long strands of DNA

How can we use bacteria to clone DNA?

• If we wish to sequence an unknown piece of DNA, we can clone the DNA into a plasmid and use a primer that binds to the plasmid near the cloning site.

• Treat vector with alkaline phosphatase - doesn’t self ligate - removes 5’ terminal phosphate which is required for ligase

• Now have molecular clone/ gene clone- grow bacteria→ millions of copies

How is automated Sanger sequencing the same as manual?

Require template DNA, primer, ddNTPs, dNTPs, DNA polymerase, means of separating reaction products by size (gel), need for reaction products detection method

How is automated Sanger sequencing different to manual?

• Usually use of thermal cycling to obtain more copies of reaction products

• Use of fluorescently labelled dideoxynucleotides so that only one reaction is necessary instead of four

• Separation using liquid gel in a capillary and automated readout from a laser scanner.