Question

Which ONE sequence of events is correct in 454 sequencing?

a. DNA extraction, amplification of exons, library preparation, sequencing, analysis of results.

b. DNA extraction, amplification of exons, sequencing, library preparation, analysis of results.

c. DNA extraction, sequencing, amplification of exons, , library preparation, analysis of results.

Answer 1

The 454 method of gene sequencing is a method for next generation sequencing. It is a much cheaper and faster technique of sequencing.

The correct sequence of events is

DNA extraction, amplification of exons, library preparation, sequencing and analysis.

So, option A is the correct answer.

Next generation sequencing mainly involves three basic steps. These are library preparation, sequencing and analysis.

The process is as follows:

The DNA sample is extracted and using restriction enzymes, these are broken into tiny fragments.

Short DNA sequences known as ADAPTORS are added to these fragments

Resin beads that contain strands complementary to that of adaptors are added to the sample; therefore the DNA fragments bind to the beads, ideally one fragment to each bead.

When beads are added, double stranded DNA become single stranded.

Numerous copies of these DNA fragments are then made.

After amplification, a gene library is prepared.

Beads are then filtered to remove unbound beads as well as those that have bound to multiple DNA fragments.

Nucleotide bases are added to this and binding of each nucleotide to the single stranded DNA is recorded using intensity of light emitted during the binding of each nucleotide.

Using this, DNA sequencing is done.

Finally the sequence is analysed.