34
Chromosome, Microdissection and Microcloning
with
restriction
enzyme,
Eco
RI.
The
smaller fragments generated (estimated
to be 3–4 kb) were ligated to a
λ
-phage
vector molecule, a reaction that is not
dependent on large quantities of insert
DNA and thus could be applied to micro-
cloning. The biochemical steps necessary
for cloning (phenol–chloroform extrac-
tion, restriction enzyme digestion, ligation
to cleaved vector) were performed in aque-
ous drops under oil. The small quantity of
recombinant DNA was then added to the
packaging mixture and the recombinant
phages were replicated in
E. coli
.U
s
ing
this technique (microdissection and mi-
crocloning), genomic clones were obtained
from
Drosophila
melanogaster
polytene
chromosomes,
D. hydei
lampbrush DNA
loops, and mouse and human metaphase
chromosomes. In these studies, the gener-
ation of multiple clones from speciFc ge-
netic regions has expedited the identiFca-
tion and isolation of a number of important
genes (e.g. the t complex in mice and the
Kruppel gene in
Drosophila
). The micro-
cloning method requires a large number
of fragments (
100) to provide sufFcient
starting material for cloning. However, the
library of recombinant clones generated
with this method usually contains DNA
inserts (
1 kb) that represent only a small
fraction (
<
5%) of the total DNA sequences
in the microdissected region.
In addition to low cloning efFciency,
the microcloning method has a number
of other problems. The use of a phage
DNA vector often results in self-religation
of phage DNA and the presence of sponta-
neous phage mutants (false positives). This
problem can be minimized by increasing
the ratio of insert to cloning vector and
by using highly puriFed phage vectors.
Reducing the initial ligation reaction vol-
ume to a few nanoliters would increase the
cloning efFciency.
To increase the number of recombinant
DNA clones, it is necessary to monitor
carefully conditions that promote DNA
degradation (e.g. excessive acid Fxation
of chromosomes during preparation of
spreads, chromosome aging on slides be-
fore staining). Chromosomes should be
prepared immediately before microdissec-
tion. All slides containing chromosome
spreads, handling pipettes, and solutions
must be thoroughly cleaned and sterilized.
Slides should be examined for the pres-
ence of contaminating microorganisms
before use.
4.1.2
Linker Adapter with PCR
Amplifcation
In the second method that involves ligation
of microdissected chromosomal DNA with
a plasmid vector or a linker adapter and
PCR ampliFcation, the chromosomal DNA
is Frst extracted with phenol–chloroform
and digested with proteinase K in nano-
liter drops. Genomic DNA is digested
with an enzyme such as
Rsa
1(or
Hpa
II)
that generates fragments of average length
of an optimal size for PCR ampliFcation
(1000–2000 bp). Another advantage of us-
ing
Rsa
1 enzyme is that its recognition
site occurs infrequently in DNA repeat se-
quences, thus minimizing the preferential
cloning of repeat sequences. Next, DNA
i
sl
ig
a
t
edtoap
l
a
sm
idve
c
to
r(
e
.g
.pUC
18) and then enzymatically ampliFed, us-
ing in the vector universal primer binding
sites that flank the insert DNA cloning
site. These primers, the M13 forward and
reverse primers, are as follows:
M13 forward sequencing primer:
5
0
-ACTGGCCGTCGTTTTAC-3
0
M13 reverse sequencing primer:
3
0
-GTCCTTTGTCGATACTG-5
0
previous page 1354 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1356 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off