Question

Explain how DNA sequencing can be automated by replacing radioactive labels
with fluorescent labels (p. 222 – 223) (Tablet p.250 – 251)
e. Explain the use of next-generation sequencing (pyrosequencing) (

Answer 1

An automated sequencing process can be set up by using primers labelled with flurocent labels instead of ddNTP with radioactive label.

When sequencing is performed with autoradiography, band of different sizes will appear in the same color regardless of whichever ddNTP is incorporated. However in an automated sequencing method the bands will fluoresce a different color for each of the four different nucleotides.

Therefore, the sequencing reaction can be carried out in one tube and as each ddNTP fluoresces in a different color, identifying the nucleotide in the sequence is easier and faster.

During a sequencing run, once the PCR with fluroscently labelled ddNTP is completed, it is placed in a gel tray and an electric current is applied so that DNA from the tray will migrate into 96 microcapillaries. As the DNA migrates towards the positive electrode, a laser will detect the wavelength of fluorescence as the DNA passes through, and is recorded by a computer. By this way the complete sequence of DNA is obtained.

e) next-generation sequencing techniques make use of the sequencing by synthesis principle instead of the chain termination method.

this technique popularized by 454 sequecning and pyrosequencing are gaining popularity due to thier high effeciency.

Although these techniques provides reads that are 400 to 500 bases long, they rarely carry any errors and hence have been used in mRNA and small RNA sequencing. they are also effectively used in whole genome sequencing and for metagenomics studies.