DNA Sequencing

Genetic Engineering

• Problem – getting enough raw material– Old solution: “bucket biochemistry”– New solution: Polymerase Chain

Reaction

PCR

• PCR is the cloning of DNA (amplification). • Copies are made and the amount of DNA

can be rapidly increased. Useful if the source of DNA is small.

• Temperature is used instead of enzymes like helicases (95oC ).

• DNA polymerase is thermostable to protect it against the reaction temperatures.

• This is an automated process and can produce sufficient DNA in 20 cycles.

PCR

PCR

DNA Sequencing• Concept: If we know the distance of each type of base from

a known origin,then it is possible to deduce the sequence of the DNA.

• For example, if we knew that there was an:

• A at positions 2, 3, 11, 13 ... and• G at positions 1, 12, ... and• C at positions 6, 7, 8, 10, 15... and• T at positions 4, 5, 9, 14....

• then we can reconstruct the sequence

DNA Sequencing• Obtaining this information is conceptually quite

simple. The idea is to cause a termination of a growing DNA chain at a known base (A,G,C or T) and at a known location in the DNA

• In practice, chain termination is caused by the inclusion of a small amount of a single dideoxynucleotide base in the mixture of all four normal bases (e.g. dATP, dTTP, dCTP, dGTP and ddATP). The small amount of ddATP would cause chain termination whenever it would be incorporated into the DNA. The incorporation of ddATP would be random and thus all possible chains that end in 'A' will exist.

DNA Sequencing

DNA Sequencing

DNA Sequencing

The separation of the sequencing fragments

DNA Sequencing

Snapshots of the detection of the fragments on the sequencer

four-dye system single-dye system

Gel Electrophoresis

• DNA is “cut” with a restriction enzyme• Sample of fragmented DNA is placed in

one of the wells on the gel. • An electrical current is passed across the

gel. • Fragment separation is based on charge

and size. • Large fragments move slowly. • Negative fragments are moved to the

right.

Gel Electrophoresis

Gel Electrophoresis• This diagram shows the

separation of 6 separate mixtures of DNA.

• The dark bands to the left are those with a large molecular mass or a positive charge

• The top mixture contains 5 fragments of DNA. Each bands corresponds to a group of DNA molecules of the same size and charge.

• The 2nd and 5th samples have the same bands. They are identical.

+-

DNA Profiling

• The technique can be used in:• Forensic crime investigations • Parentage Issues • Animal breeding pedigrees • Disease detection

Fingerprinting