Chromosome, Microdissection and Microcloning
37
structure and are composed of two tan-
domly arranged halves.
Alu
repeats are
approximately 300 bp long and are present
at a copy number in excess of 10
6
per
haploid human genome equivalent. The
L1 repeats, however, are present in 10
4
to
10
5
copies per genome.
Alu
and L1 repeats
occur preferentially in light Giemsa bands
and dark Giemsa bands respectively.
Oligonucleotide primers based on hu-
man
Alu
sequences and L1 sequences
were designed and used in PCR ampli-
Fcation of human DNA sequences from
hamster–human hybrid cells containing a
single human chromosome. The following
primers were utilized:
Alu
-559: 5
0
-AAGTCGCGGCCGCTTGCA-
GTGAGCCGAGAT-3
0
Alu
-517: 5
0
-CGACCTCGAGATCT(C/T)-
(G/A)GCTCACTGCAA-3
0
L1Hs: 5
0
-CATGGCACATGTATACATA-
TGTAAC(T/A)AACC-3
0
A drawback of direct ampliFcation using
degenerate
primers,
however,
is
that
minute quantities of any contaminant
DNA will also be ampliFed along with
the chromosomal DNA. Therefore, certain
precautions are necessary. Chromosome
spreads should be dropped on clean, sterile
coverslips. All solutions and even the
oil used in the microdissection system
should be Flter-sterilized and placed under
UV light for several hours or overnight
before use.
Also, glass cutting needles should be
acid/alcohol washed and sterilized under
UV light. Pipettes should be designated
for use in chromosome fragment ampli-
Fcation only. Occasionally, the
Thermus
aquaticus
DNA polymerase used in PCR
reactions becomes contaminated with for-
eign DNA (bacterial or yeast) that served
as the PCR template. Therefore, the lots
of enzyme should be tested before use
in microdissection experiments. No PCR
product should be detectable after three
consecutive rounds of ampliFcation in the
absence of template DNA. ±inally, it is cus-
tomary to run a ‘‘water control’’ (reaction
without DNA template) in tandem with
each PCR ampliFcation to ensure that the
test reaction DNA product was generated
from chromosomal fragments.
Direct ampliFcation of microdissected
chromosomal DNA also has unique prob-
lems. Often, one or more ampliFed prod-
ucts cannot be visualized on agarose
gel after one round of PCR cycle. This
situation can be rectiFed, however, by
increasing the number of chromosome
fragments in the PCR reaction and in-
creasing the primer concentration (
1
µ
g).
More important, decreasing the annealing
temperature of the initial thermal cycles of
PCR (27–30
C) was found to enhance the
ampliFcation product. Running an addi-
tional round or two of PCR ampliFcation
is recommended. This is accomplished by
removing an aliquot (1/10th) from the Frst
round and repeating the PCR. Chromoso-
mal DNA that is precut with restriction
enzyme (e.g.
Hpa
II) would generate a
homogeneous population of fragments,
amenable to uniform PCR ampliFcation.
A more troublesome problem – the isola-
tion of large number of clones containing
repeat sequences – is the result of the
combination of an abundance of these se-
quences throughout the human genome
and the low stringency of PCR annealing
temperatures. The problem can be mini-
mized (but not avoided) by Fne-tuning the
annealing temperature in the initial PCR
cycles to a point that biases the PCR to-
ward ampliFcation of unique sequences.
Alternatively, following the generation of
PCR product it may be useful to perform
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