36
Chromosome, Microdissection and Microcloning
the
Drosophila
polytene X chromosome us-
ing degenerate ‘‘universal’’ primers. More
recently, libraries of speciFc human chro-
mosome bands were constructed using
universal primers and PCR. The use of
Alu
-speciFc primers to screen for hu-
man genes in complex DNA mixtures has
been described. Probes generated from
microdissected chromosomal DNA frag-
ments with
Alu
-PCR have been used to
localize speciFc chromosomal sequences
in YAC recombinant clones. They were
also used to generate sequence-tagged
sites (STSs) from discrete chromosomal
regions. Direct PCR ampliFcation of mi-
crodissected DNA has a few advantages
over the methods that employ cloning Frst.
Direct ampliFcation of picogram amounts
of DNA obviates the time-consuming steps
that involve restriction enzyme digestion,
ligation, and subcloning. This approach
minimizes the loss of minute starting
quantities of DNA during the various ma-
nipulations.
Most important, the clones constituting
the Fnal library contain chromosome-
speciFc DNA inserts of considerably larger
size, depending primarily on the recom-
binant library that was screened. This
method does not involve cloning; hence
the size of the library depends entirely on
the efFciency and completeness of PCR
ampliFcation.
The completeness of the PCR product
is dependent on the sequence of primers
used and the PCR reaction conditions (e.g.
annealing temperatures during the early
cycles, primer extension times), concentra-
tion of deoxynucleoside triphosphates, the
quality of DNA polymerase, and Fnally the
number of cycles (usually 60–90) required
to amplify sufFcient amounts of DNA in-
serts containing unique sequences.
The term
universal
primers
refers to
a mixture of oligonucleotide sequences
that lack absolute complementarity to the
target template sequences. They contain
multiple degenerate bases. When one is
using these primers in PCR ampliFcation
and selecting lower PCR annealing tem-
peratures (46–48
C), complementary se-
quences that contain signiFcant homolo-
gies can be ampliFed. ±or example, the
following primer was recently used to
generate a probe library from
Drosophila
polytene chromosome fragments:
5
0
-TTGCGGCCGCATTNNNNTTC-3
0
With complete degeneracy at positions 4
to 7 from the 3
0
end, authentic DNA
sequences from the
Drosophila
genome
were isolated. A similar primer has been
described in PCR ampliFcation of DNA
from human chromosomes. This 22-mer,
however, contains complete degeneracy at
6 nucleotides (positions 11–16), with the
following sequence:
5
0
-CCGACTCGAGNNNNNNATGTGG-3
0
In general, a nonspeciFc primer should
have the following structure from 5
0
to
3
0
ends: two or more protective (cap) nu-
cleotides (e.g. TT and CCGA in the primers
just described), four to six nucleotides
with complete degeneracy, and Fnally, a
terminal 3
0
sequence that occurs at high
frequency in the genome of the species
in which DNA is ampliFed, such as TTC
and ATGTGG in the
Drosophila
and hu-
man genomes respectively. ±urthermore,
a restriction endonuclease site (e.g.
Not
I
and
Xho
I, in the just-described primers
respectively) may be added for easy recog-
nition of the PCR product and subsequent
cloning in an appropriate vector. The
Alu
repeats (SINEs) and L1 repeats (LINEs) are
short interspersed repeated DNA elements
distributed throughout the genomes of
primates.
Alu
elements are dimeric in
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