DNA
SEQUENCING
Background
DNA
sequencing involves a process that you have learned
earlier, that is the Polymerase Chain Recation or
PCR.
The
purpose of sequencing is to determine the order of
the nucleotides of a gene. This order is the key to
the understanding of the human genome. Frederick Sanger
was first accredited with the invention of DNA sequencing
techniques.
Sanger's
approach involved copying DNA strands which would
show the location of the nucleotides in the strands
though the use of X-Ray machines. This technique is
very slow and tedious, usually taking many years to
sequence only a few million letters in a string of
DNA that often contain hundreds of millions or even
billions of letters. Modern techniques make use of
fluorescent tags instead of X-rays. This significantly
reduced the time required to process a given batch
of DNA
In
1991, working with Nobel laureate Hamilton Smith,
Venter's genomic research project (TIGR) created a
bold new sequencing process coined 'shotgunning.'
Golden & Lemonick (2000) describe 'shotgunning':
"Using
an ordinary kitchen blender, they would shatter the
organism's DNA into millions of small fragments, run
them through the sequencers (which can read 500 letters
at a time), then reassemble them into full genomes
using a high speed computer and novel software written
by in-house computer whiz Granger Sutton"
This
new method not only uses super fast automated machines,
but also the fluorescent detection process and the
PCR DNA copying procedure. This method is very fast
and accurate compared to older techniques.
How
DNA Sequencing Is Done
DNA
sequencing is a complex nucleotide-sequencing technique
including three identifiable steps:
·
Polymerase Chain Reaction (PCR)
· Sequencing Reaction
· Gel Electrophoresis & Computer Processing
Chromosomes,
which range in size from 50 million to 250 million
bases, must first be broken into much shorter pieces
( PCR step)
Each
short piece is used as a template to generate a set
of fragments that differ in length from each other
by a single base that will be indentified in a later
step (template preparation and sequencing reaction
steps)
The
fragments in a set are separated by gel electrophoresis
(separation step).
New fluorescent dyes allow separation of all four
fragments in a single lane on the gel.
Picture
reproduced with permission from http://allserv.rug.ac.be/~avierstr/principles/seq.html
The
final base at the end of each fragment is identified
(base-calling step). This process recreates the original
sequence of As, Ts, Cs, and Gs for each short piece
generated in the first step
Current electrophoresis limits are about 500 to 700
bases sequenced per read. Automated sequencers analyze
the resulting electropherograms and the output is
a four-color chromatogram showing peaks that represent
each of the 4 DNA bases.
The fluorescently labelled fragments that migrate
through the gel, are passed through a laser beam at
the bottom of the gel. The laser exits the fluorescent
molecule, which sends out light of a distinct color.
That light is collected and focused by lenses into
a spectrograph. Based on the wavelength, the spectrograph
separates the light across a CCD camera (charge coupled
device). Each base has its own color, so the sequencer
can detect the order of the bases in the sequenced
gene.
Picture
reproduced with permission from http://allserv.rug.ac.be/~avierstr/principles/seq.html
After
the bases are "read," computers are used
to assemble the short sequences (in blocks of about
500 bases each, called the read length) into long
continuous stretches that are analyzed for errors,
gene-coding regions, and other characteristics.
