Lecture 207 of 373: Pyrosequencing; Shotgun and Clone Contig Approach; Chromosome Walking (21 mins) | CUET (Common University Entrance Test) PG Zoology (SCQP28) | Complete Video Course 373 Lectures [222 hrs : 42 mins]

Details

Pyrosequencing

Pyrosequencing, like the chain termination method, requires a preparation of identical single-stranded DNA molecules as the starting material.

These are obtained by alkali denaturation of PCR products or, more rarely, recombinant plasmid molecules. After attachment of the primer, the template is copied by a DNA polymerase in a straight forward manner without added dideoxynucleotides.

As the new strand is being made, the order in which the deoxynucleotides are incorporated is detected, so the sequence can be “read” as the reaction proceeds.

The addition of a deoxynucleotide to the end of the growing strand is detectable because it is accompanied by release of a molecule of pyrophosphate, which can be converted by the enzyme sulfurylase into a flash of chemiluminescence.

Shotgun approach

The shotgun approach, in which the genome is randomly broken into short fragments. The resulting sequences are examined for overlaps and these are used to build up the contiguous genome sequence.

Clone contig approach

The clone contig approach, which involves a pre-sequencing phase during which a series of overlapping clones is identified. This contiguous series is called a contig. Each piece of cloned DNA is then sequenced, and this sequence placed at its appropriate position on the contig map in order to gradually build up the overlapping genome sequence

Chromosome Walking

One technique that can be used to assemble a clone contig is chromosome walking. To begin a chromosome walk a clone is selected at random from the library, labelled, and used as a hybridization probe against all the other clones in the library.

Those clones that give hybridization signals are ones that overlap with the probe. One of these overlapping clones is now labelled and a second round of probing carried out. More hybridization signals are seen, some of these indicating additional overlaps.

Gradually the clone contig is built up in a step-by-step fashion. But this is a laborious process and is only attempted when the contig is for a short chromosome and so involves relatively few clones, or when the aim is to close one or more small gaps between contigs that have been built up by more rapid method.