Gene Cloning

124 Gene Cloning
(a)
Labeled single-stranded
DNA probe
Single-stranded
target DNA
(b)
Figure 5.4 Hybridization of DNA probes. a) The DNA to be probed is
denatured to make it single-stranded and immobilized on a solid support; a
labeled DNA probe is added. b) The probe will anneal to molecules with a
complementary sequence of bases, hydrogen bonds will form between
complementary bases and a double-stranded duplex will be formed. This process
is often referred to as hybridization. In this way molecules complementary to
probe will be specifically labelled and hence can be identified.
region, either regulatory sequences or adjacent genes. Alternatively, you
might know the sequence of the gene in a related organism; DNA hybridization
using a probe derived from one species can be used to detect homologous
genes either in the same organism or in other species. If you are trying
to clone a gene for which there is no sequence data it may still be possible
to use DNA hybridization, as long as you can purify the protein product of
the gene of interest. If you can determine the sequence of part, usually the
N-terminus, of the protein you can then derive the DNA sequence from this.
5.6 Oligonucleotide Probes
Short synthetic DNA molecules similar to those used as primers in PCR and
sequencing can be used as probes in hybridization procedures.
Oligonucleotide probes can be designed from known DNA sequence but
more usually are derived from amino acid sequence. Relatively small samples,
containing as little as 5 pmol of purified protein, can be sequenced by
the Edman degradation. This is a sequential procedure in which the N-
terminal amino acid is chemically cleaved from the protein and identified
by liquid chromatography; the procedure is repeated with subsequent
amino acids. This is now an automated process; it can reliably identify the
first 20 amino acids of a protein, although some proteins are blocked making
sequencing by this approach very difficult.
Q5.5.Why is it not possible to derive an unambiguous DNA sequence from
the amino acid sequence of a protein?