Biology Reference
In-Depth Information
7.16 Metagenomics 290
7.17 Proteomics: Another “-Omic”
291
7.18 Functional Genomics 292
7.19 Structural Genomics—Another New Horizon?
293
7.20 Comparative Genomics 294
7.21 Interactomes or Reactomes 295
7.22 The Post-Genomic Era: Systems Genetics
295
General References
297
References Cited
297
7.1 Overview
Sequencing, resolving the order of the bases in DNA, is carried out on genomic
DNA, cDNA, or mitochondrial DNA. Sequencing is a first step in evaluating reg-
ulatory sequences as well as coding and noncoding regions. DNA sequences
are used to reconstruct phylogenies, identify taxonomic groups, evaluate
the evolution of genomes, and conduct research on population ecology and
genetics.
There are two original methods for sequencing DNA (first-generation meth-
ods). For both, sequencing involves four procedures: 1) cloning/preparing tem-
plate DNA, 2) performing the sequencing reactions, 3) electrophoresis of the
samples, and 4) compiling and interpreting the data.
The most commonly used first-generation sequencing method, the Sanger or
dideoxy chain-terminating protocol, was developed in 1975. The Sanger method
involves synthesis of DNA in vitro on a single-stranded template by using a
primer, a mixture of labeled deoxynucleotide triphosphates (dNTPs), and dide-
oxynucleotide triphosphates (ddNTPs). The reaction terminates at the position at
which the ddNTP, instead of the dNTP, incorporates into the growing DNA chain.
Four different reactions are carried out, one for each base. The DNA fragments
from the four reactions originally were separated by acrylamide gel electropho-
resis with the base sequence being identified by autoradiography of the four
banding patterns.
In the other first-generation method, a “chemical” sequencing method devel-
oped by Maxam and Gilbert in 1977, single-stranded DNA derived from double-
stranded DNA and labeled at the 5 end is subjected to cleavage protocols that
selectively make breaks on one side of a specific base. Fragments from the reac-
tions were then separated according to size by acrylamide gel electrophoresis,
and the sequences are identified by autoradiography.
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