Biology Reference
In-Depth Information
opportunities became possible only after DNA sequencing methods were devel-
oped in the late 1970s.
Two basic DNA sequencing methods were developed about the same time:
the chemical, or Maxam-Gilbert method (1977, 1980), and the chain-termination
method of
Sanger et al. (1977)
. Both used the same basic approaches: 1) cloning
or preparing the DNA templates, 2) performing the sequencing reactions on the
DNA templates, 3) electrophoresis of the samples, and 4) compiling and inter-
preting the data.
DNA to be sequenced can be genomic DNA, mitochondrial DNA, or cDNA.
Because cDNA lacks introns and regulatory elements, sequencing of cDNA pro-
vides less information than genomic DNA. Sequencing only cDNA probably
would miss some genes that are expressed at very low levels or in a tissue- or
time-dependent manner.
Effective computer tools are needed to discover the sequences that actu-
ally code for a gene, because up to 90% of genomic sequences can be noncod-
ing DNA (that do not code for proteins). Some noncoding DNA sequences are
associated with centromeres or telomeres, and others have no known function.
Different computer programs have been developed to search DNA sequence
data and identify possible regulatory sequences, potential start or stop codons,
open reading frames (ORFs), and sequences that may indicate the location of
intervening sequences or introns. Unfortunately, no computer program is 100%
accurate in identifying genes (
Bork 2000
).
The most reliable way to find genes currently is to identify them because they
are similar to known proteins from the same or other organisms or similar to
cDNAs from the same or a closely related organism (
Stormo 2000
). However,
many genes have no significant similarity with other known sequences (
orphan
genes
). Furthermore, just because a DNA sequence is similar to the sequence of
a known gene in
D. melanogaster
does not necessarily indicate that this gene
functions in the same manner.
The length of DNA that can be sequenced by a single Sanger reaction var-
ies from 200 to
≈
1000 bases, depending upon the method used (
Sambrook
and Russell 2001
). Vectors can contain DNA inserts ranging in size from 100 to
1,000,000bp. For example, yeast artificial chromosomes (YACs) can contain
inserts up to 1 million base pairs, and cosmids can contain inserts of 30,000-
45,000 bp. Thus, cloned DNA typically is converted into smaller segments or
sub-
clones
that are then inserted into vectors that are specialized for sequencing,
such as M13 or plasmid sequencing vectors.
