Biology Reference
In-Depth Information
Single-stranded probes should not anneal to themselves so that hybrids com-
posed of reannealed probes cannot be made. RNA probes do not need to be
denatured before being used because they are already single-stranded. Double-
stranded DNA probes must be denatured before using, which produces two
strands. If only one of the DNA strands has been labeled, the unlabeled strand
can dilute the reaction mixture.
Single-stranded probes are prepared from DNA templates by synthesizing
labeled DNA that is complementary to sequences cloned in a bacteriophage
vector such as M13 or a phagemid. RNA probes can be produced by transcrip-
tion of ds DNA in a vector with a powerful promoter derived from
E. coli
bac-
teriophages T7 and T3 by a bacteriophage DNA-dependent RNA polymerase.
The labeled transcript produced is complementary to one of the two template
strands. The probe can therefore be used as strand-specific probes in hybridiza-
tion reactions. cDNA probes are used to isolate cDNA clones of genes that are
expressed in specific cells or tissues.
6.7.5 Synthetic Probes
The knowledge of the sequence of a few amino acids in a protein will allow
a specific gene to be isolated with a synthetic probe. Automated machines can
synthesize short segments of ss DNA in which the sequences are defined pre-
cisely. Probes also can be produced that consist of alternative sequences, as
determined by the degeneracy of the genetic code. Because there are 64 pos-
sible codons and only 20 amino acids, most amino acids are coded for by
more than one codon. Thus, a probe that consists of a mixture of degenerate
sequences can be used to screen libraries.
6.8 Baculovirus Vectors Express Foreign Polypeptides in Insect Cells
Escherichia coli
, infected with plasmid or phage expression vectors, has been
used to express foreign eukaryotic genes. However, biologically active proteins
are difficult to produce in
E. coli
because
E. coli
cannot make posttranslational
changes such as
glycosylation
and
phosphorylation
, processes that are essential
to effective protein function in eukaryotes. As a result, eukaryotic expression
vectors have been developed for use in yeast and insect cells.
The most effective expression vectors used in insect cells were engineered
from baculoviruses (
Luckow and Summers 1988, Maiorella et al. 1988, Jarvis et al.
1990
, O'Reilly et al. 1992,
van Oers 2011
).
Baculoviruses
are viruses with ds, circu-
lar DNA genomes contained within a rod-shaped protein coat. The Baculoviridae
are divided into three subgroups: nuclear polyhedrosis viruses (NPVs), granulosis
