Biology Reference
In-Depth Information
that purpose are available now. Once RNA has been isolated, it must be evalu-
ated for quality, often by agarose gel electrophoresis.
6.5 Enzymes Used in Molecular Biology Experiments
Many enzymes used in genetic engineering have been mentioned in this and
previous chapters.
Table 6.1
summarizes their names, principle activity(ies),
sources, and functions in genetic manipulations. Enzymes used to synthesize
DNA include terminal transferase, DNA polymerase I, and reverse transcriptases.
Enzymes that modify DNA include S1 nuclease, exonuclease III, Bal31 nuclease,
and DNase I. There are
>
1400 restriction endonucleases that can cleave DNA in
a predictable manner. T4 and
E. coli
DNA ligases join DNA molecules. Calf intes-
tinal phosphatase (CIP) and T4 polynucleotide kinase are used to modify the 5
′
ends of DNA molecules.
6.6 Isolating a Specific Gene from a Library if Whole-Genome
Sequencing is Not Done
The production of a library is only a first step. Because the cost of sequencing
whole genomes has decreased dramatically, it is now possible to screen librar-
ies by sequencing them (see Chapter 7). However, if whole-genome sequencing
is not done, the information in a library can only be obtained if the library can
be screened. Screening identifies specific genes and provides information about
genome organization, or information about gene regulation. The ability to
screen a library is dependent upon the availability of a
probe
. As pointed out in
Chapter 5, a probe is a molecule that can be labeled with radioactive isotopes or
by nonradioactive methods.
There are four ways to obtain a suitable probe for your library. 1) The amino-
acid sequence of the protein is known for the species being studied, or a related
species, and can be used to predict and synthesize the sequence of an oligonu-
cleotide (oligo) hybridization probe. Because the genetic code is degenerate,
the probe used may actually incorporate a mixture of oligos with optional bases,
especially in the third site of the codon. 2) The gene of interest has already
been cloned from a related organism, so that it can be used as a heterologous
hybridization probe. For some genes, particularly housekeeping genes, conser-
vation of functional domains in proteins has been extensive, so that probes from
other species can be used effectively. 3) The protein is abundant in a particu-
lar tissue so the relevant clone can be identified by its relative abundance in a
tissue-specific cDNA library. 4) If the protein has been purified, it can be used to
generate an antibody against it. The antibody can be used to identify recombi-
nant cells for the presence of the specific enzymes.
