Biology Reference
In-Depth Information
Metagenomics analysis can be done using several methods. One method
involves extracting genomic DNA from organisms from a sample from a specific
environment. The DNA is then cut into uniform lengths and inserted into a vec-
tor, replicated in
E. coli
, and sequenced using traditional Sanger sequencing. The
clones are then sequenced and analyzed. More recently, sequencing is by “shot-
gun sequencing” of randomly sampled DNA, which is less expensive, using next-
generation sequencing methods.
Data analysis involves assembly of short overlapping sequence reads into a
consensus sequence and predicting which genes or partial genes are present. In
complex communities with many microbial species, the reads may not assemble
because overlapping reads may not be produced. The amount of an individ-
ual microbe's genome that is sequenced depends on how abundant it is in the
environment and how many reads are produced. It also is possible to reverse-
transcribe RNAs from the environment into DNA and sequence these, which pro-
vides information on metabolic pathways.
7.17 Proteomics: Another “-Omic”
Once we know the complete DNA sequences of organisms (genomics), the next
goal is to understand how the genes are translated in living cells (proteomics).
What proteins function to provide structure and function in the living organ-
ism? Proteomics was first formalized as a term in 1996 and combines “proteins”
and “genomics.” Definitions of proteomics and the other “-Omics” seem to be
evolving, however.
Proteomics
is the genome-wide analysis of proteins and includes three
aspects: 1) characterization of proteins and their posttranslational modifica-
tions, 2) “differential display” to compare protein levels and types, and 3) stud-
ies of protein-protein interactions. Proteomics uses mass spectrometry and
two-dimensional protein gel electrophoresis (
Geisow 1998, Dutt and Lee 2000,
Pandey and Mann 2000
).
Two-dimensional gel electrophoresis allows the identification of proteins
whose expression changes in an interesting manner from that of a reference
point. Two-dimensional gel electrophoresis separates proteins
by charge
using
isoelectric focusing and
by size
using sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Up to 11,000 proteins from a single mixture can be
resolved (
Dutt and Lee 2000
). SDS-PAGE can purify proteins for amino-acid anal-
ysis, mass spectrometry, and amino-acid sequencing.
Mass spectrometry of proteins separated on two-dimensional gel electropho-
resis generates different types of structural information about a protein. For
