Unraveling the Human Thread of Life - Genome Sequencing Strategies.
The Human Genome Project (HGP) and Celera Genomics chose different strategies to determine the human genome sequence. The HGP used "hierarchical shotgun sequencing," also called the "clone-by-clone" or "BAC-to-BAC" approach. DNA sequencing was preceded by constructing a physical map, which aided the positioning of DNA segments that were sequenced. Celera Genomics took a route known as "whole-genome shotgun sequencing," which circumvented the production of a map and so was faster.
Hierarchical shotgun approach. To begin with, chromosomal DNA was cut by certain enzymes to produce overlapping fragments (100,000--200,000 base pairs long), each of which was inserted into a DNA piece called a bacterial artificial chromosome (BAC). The BACs, capable of replicating themselves in bacterial cells, provide a way to generate multiple, exact copies--or clones--of the human DNA segments.
Each BAC was cut with a specific enzyme to produce a unique pattern of fragments called a fingerprint. By comparing the patterns obtained with different cloned DNA segments, overlapping clones could be identified and arranged in order. The sites of cleavage were also used as landmarks on the genome, thus giving a physical map.
The BAC clones were then broken into smaller, overlapping fragments (each about 2,000 bp) that were inserted into bacterial viruses and amplified by replication in bacterial cells. Sequencing of these "subclones" [see "DNA Sequencing Technique," p. ---- ] gave a sequence of about 500 bp near one end of each human DNA segment. A computer program was employed to identify the overlaps and assemble the millions of sequences obtained.
Whole-genome shotgun approach. Human chromosomal DNA was randomly broken by forcing it through pressurized syringes, to generate two sets of fragments with average lengths of 2,000 bp and 10,000 bp, respectively. Each fragment was inserted in a plasmid, which is a small, circular DNA piece that can replicate in bacterial cells.
Replication of each plasmid produced many copies of the inserted human DNA. Sequencing of the cloned DNA yielded about 500 bp of sequence at each end of the human DNA insert. Computer algorithms then sought out the sequence overlaps among fragments and assembled the millions of sequences.
|Printer friendly Cite/link Email Feedback|
|Author:||Dadachanji, Dinshaw K.|
|Publication:||World and I|
|Article Type:||Brief Article|
|Date:||Sep 1, 2001|
|Previous Article:||Unraveling the Human Thread of Life - What Is a Genome Map?|
|Next Article:||Unraveling the Human Thread of Life - DNA Sequencing Technique.|