Biology, asked by Sutapa5684, 1 year ago

Discuss the sequencing techniques and the automation to complete the whole genome sequencing.

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Answered by mansabhartiy
0

Besides screening for specific DNA sequences, automated sequencing of insert ends is often performed to analyze the metagenomic libraries in large scale. For this purpose, it is advisable to use a fosmid vector with inducible copy number, in order to reduce the costs for DNA preparation.

The same principle also holds for manual sequencing of single fosmids. If the copy of the plasmid can be induced, preparation of pure DNA is easy. If a single-copy vector has been employed, we found that using the Qiagen midi prep kit containing the Top10 gravity flow columns works best for getting high-quality DNA, provided that the few protocol changes are followed with regard to those for low copy plasmids. Purifying fosmid DNA from a 60–80 ml E. coli culture was sufficient using the single-copy vector .

Sequencing of a complete single-fosmid DNA can be either done by producing a subclone library of smaller fragments in regular plasmids for Sanger sequencing or by employing direct sequencing (e.g., pyrosequencing (454) technology) on the recombinant fosmid DNA.

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