Chromosome, Microdissection and Microcloning
33
Fig. 5
Example of fragments
microdissected from human
chromosomes 3 using videomicroscopy.
MagniFcation is 3200
×
on the display
monitor. The fragments cut represent
from top to bottom bands 3p21, 3p14,
3p13, and 3q21. Each fragment
measures approximately 0.5
µ
mand
contains 10–15 Mbp.
metaphase chromosomes were modifed
to minimize DNA degradation as reviewed
earlier. When care is taken to avoid pro-
longed acid fxation, microcloning yields
recombinant clones with relatively larger
DNAin
s
e
r
t
s
.F
in
a
l
l
y
,m
e
th
o
d
ssu
cha
s
videomicroscopy were incorporated to pro-
vide higher magnifcation and resolution
o± minor chromosome bands.
To date, three distinct approaches can
be described ±or generating recombinant
DNA ±rom microdissected chromosomal
±ragments. In the frst and earlier method,
DNA was extracted ±rom chromosome
±ragments in a nanoliter aqueous drop and
digested with
Eco
RI restriction enzyme,
whereupon the digestion products were
directly cloned into a
λ
-phage vector (this
method is o±ten called
microcloning
). The
second method is an extension o± the frst
and utilizes PCR. Microdissected DNA is
frst digested with a restriction enzyme;
then, digested DNA is enzymatically lig-
ated either to a linearized plasmid vector
or to oligonucleotide linker primers. The
cloned inserts are then amplifed by PCR
using vector-specifc primers or the linker
primers. This method generates a large
number o± recombinant clones (usually
20 000). DNA ±rom a number o± human
chromosome bands o± considerable clini-
cal interest has been cloned in this way, as
discussed in Sect. 4.2. The third method
involves the direct PCR amplifcation o±
chromosomal DNA using care±ully de-
signed oligonucleotide primer sequences
that are o±ten termed
universal
or random
primers, and more recently using degen-
erate oligonucleotide primers (DOP-PCR).
The universal primers method is based on
Alu
repeat sequences that are naturally
present in the genome. The prerequi-
site o± using universal or repeat-sequence
primers is that they occur ±requently in the
genome being studied. The PCR product
is then used to probe a more complete
recombinant library (cDNA or genomic).
The library chosen ±or screening should
contain clones with large inserts [e.g. cos-
mid, bacterial/yeast artifcial chromosome
(BAC/YAC) clones]. Hence i± a PCR reac-
tion is symmetrical and unbiased, a more
complete chromosomal region–specifc li-
brary can be generated by using the PCR
product as a probe. Since our ±ocus is mi-
crodissection and microcloning, a more
detailed account o± these methods is given
in Sect. 4.2, with emphasis on the role
o± PCR and fluorescent
in situ
hybridiza-
tion (FISH).
4.1.1
Microcloning
To initiate the direct cloning o± microdis-
sected chromosomal DNA, the DNA was
extracted, frst with phenol–chloro±orm,
then with proteinase K, and then digested
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