Biology Reference
In-Depth Information
Restriction sites can be inserted at the 5
′
end of each primer so that the ampli-
fied DNA can be cloned directly into a vector after digestion of the amplified
DNA (
Kaufman and Evans 1990
). Because the restriction sites can be the same or
different in the two primers, the researcher can tailor the ends of the PCR prod-
uct to the specific vectors required for the project (
Sambrook and Russell 2001
).
Various commercial kits allow direct cloning of PCR products.
The isolation of a gene requires some prior knowledge of the gene sequence
(
Clackson et al. 1991
). If a probe (primer) is available from another species,
genomic DNA can be screened by the PCR using standard or degenerate primers
(
McPherson et al. 1991, Clackson et al. 1991
). This success of this approach was illus-
trated by the cloning of a sodium channel gene from
Drosophila
and the house
fly,
Musca domestica
, using degenerate primers (
Knipple et al. 1991
). Several ver-
tebrate sodium channel genes had been cloned and comparisons of the inferred
amino-acid sequences of the alpha subunits of sodium channels from rat brain and
rat skeletal muscle to that of the electric eel revealed a 70% homology when con-
servative substitutions were taken into account. Two sodium channel genes (
para
and DSC1) cloned from
Drosophila
were homologous to the vertebrate sodium
channel genes. Using this information, it was possible to generate DNA primers to
isolate a segment of the gene homologous to
para
from the house fly.
The PCR was performed on genomic house fly DNA using degenerate prim-
ers. The 5
′
-end primer consisted of a 256-fold degenerate sequence 20nt long.
The 3
′
-end primer consisted of a 64-fold degenerate sequence 21 nt long. (When
using degenerate primers, the primer length may be shorter than the typical
25-30 nt.) Both had additional sequences appended to their 5
′
ends to provide a
Hind
III and
Xba
I restriction-enzyme recognition sequence, respectively, to facili-
tate cloning the amplification products. The PCR product was 104bp, consisting
of 87 bp of coding sequence plus the flanking sequences attached to the 5
′
ends
of the primers. To confirm that the PCR-generated DNA was derived from the
house fly, amplified DNA was labeled with
32
P and used as a probe of genomic
Southern blots containing digests of house fly,
Drosophila
, and mouse DNA. The
only specific hybridization signal after high-stringency washing was to the fly
DNA. The PCR products were cloned and sequenced, and the sequence isolated
from the house fly differed from that of
Drosophila
at only 16 nucleotides (81.6%
similarity). The substitutions, primarily in the third base of the codon, had no
effect on the amino-acid sequence.
Doyle and Knipple (1991)
subsequently used the same degenerate primers
to amplify DNA from seven insects and an arachnid, including the tobacco bud-
worm,
Heliothis virescens
;
Aedes aegypti
; the diamondback moth,
Plutella xylo-
stella
; the gypsy moth,
Lymantria dispar
; the cabbage looper,
Trichoplusia ni
; the
