Chromosome, Microdissection and Microcloning
35
The cloned DNA is amplifed using PCR
with M13 primers. ThereFore, only one pair
oF oligonucleotide primers is used For the
simultaneous amplifcation oF many diF-
Ferent DNA inserts. PCR amplifcation is
perFormed according to the Following ther-
mal profle: 25 one-minute cycles oF denat-
uration at 94
C (frst cycle, 5 min), 1 min
oF annealing at 55
C, and 2 min oF primer
extension at 72
C. ±ollowing the initial
amplifcation, the DNA inserts are cleaved
with a second enzyme (
Eco
RI) that flanks
the
Rsa
1 site. The inserts are subsequently
subcloned into a second plasmid vector
and propagated in the appropriate bacterial
host to generate the band-specifc recombi-
nant minilibrary. This procedure was frst
employed to clone specifc genes From hu-
man chromosome 8 From Langer–Giedion
syndrome patients. This cloning method
oFFers several advantages. Cloning in plas-
mid vectors is easier than cloning in
phages and can be more rapidly charac-
terized. Also, the smaller size oF the vector
renders DNA inserts in plasmid vectors
more amenable to enzymatic amplifca-
tion and direct sequencing than they are
in phage vectors. The introduction oF PCR
allows the amplifcation oF a population
oF clones that would otherwise go unde-
tected. ±inally, the use oF PCR signifcantly
reduces the number oF chromosomal Frag-
ments initially required For cloning.
A similar method in which the mi-
crodissected DNA was ligated to a linker
adapter molecule instead oF a plasmid vec-
tor Followed by PCR amplifcation was
described. The linker adapter consisted oF
a 24-nucleotide molecule with a terminus
complementary to the
Mbo
1enzymerecog-
nition sequence. The
Mbo
1linkeradapters
have the Following sequence:
5
0
-GATCTGTACTGCACCAGCAAATCC-3
0
3
0
-ACATGACGTGGTCGTTTAGG-5
0
The linker adapter is Formed by anneal-
ing a mixture containing 100
µ
gmL
1
oF each oligonucleotide at 58
CFo
r1h
.
Prior to the annealing step, the 24-mer
oligonucleotide component carrying the
5
0
Mbo
1 complementary overhanging end
is phosphorylated. This step is necessary
to prevent the Formation oF tandem ar-
rays oF linkers during ligation, which may
interFere with the amplifcation oF chro-
mosomal DNA. ±ollowing chromosomal
DNA extraction and digestion as described
earlier, the DNA is ligated to the
Mbo
1
linker adapter at 19
C For 16 h. PCR
amplifcation is carried out as Follows:
35 one-minute cycles oF denaturation at
94
C, primer annealing (2 min, 52
C),
and primer extension (3 min, 73
C). The
amplifed DNA is subsequently cloned
into
Bam
HI linearized Bluescript plas-
mids aFter extraction and gel purifcation.
The library is then propagated in compe-
tent bacteria.
4.1.3
PCR Amplifcation oF Chromosomal
DNA
The third method oF microcloning em-
ploys PCR amplifcation oF microdissected
chromosomal DNA Fragments without
prior manipulation. The PCR reaction
product is examined For selF-completeness,
is radioisotopically labeled, and is used
to probe a more complete recombinant
library. In this direct method, DNA se-
quences From a specifc chromosomal
region are isolated by amplifcation oF
the microdissected DNA with PCR, us-
ing a set oF universal primers containing
a series oF degenerate base sequences.
The amplifcation product would repre-
sent a heterogeneous population oF DNA
sequences. Direct PCR amplifcation oF
microdissected DNA has been recently
employed to isolate genes From band 2± oF
previous page 1355 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 1357 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off