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The biological basis of DNA sequencing technology

Virtually all modern DNA sequencing, regardless of whether it is done on an automated sequencer or by autoradiography, relies on the Sanger method of DNA replication with dideoxy chain termination and separation of the resulting molecules by polyacrylamide gel electrophoresis.

In simple terms, this means that in order to be sequenced, a DNA molecule must be copied in a test tube (in vitro ) by a DNA polymerase enzyme.

The DNA fragment to be sequenced must first be cloned into a vector (such as a plasmid) to obtain a sufficient quantity of the fragment to be sequenced.

Another limitation imposed by the technology is that sequences can only be determined in approximately 500 base pair (bp) chunks known as "reads".

This is due to both the biochemistry of the DNA polymerase reaction and the resolution of polyacrylamide gel electrophoresis.

Yet most genes contain many thousands of bp and many modern sequencing projects are intended to produce complete sequences of large genomic regions or even entire chromosomes (many millions of bp).

As a result, all sequencing projects must involve the division of the target DNA into a set of overlapping 500 bp fragments.

Once the sequences of these fragments are determined, they must be pieced back together into "contigs" (lab slang for "contiguous sequenced regions") by computer "assembly" programs.

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Using Computers for Molecular Biology
Stuart M. Brown, Ph.D., RCR, NYU Medical Center

Comments to: browns02@mcrcr.med.nyu.edu