38
Chromosome, Microdissection and Microcloning
suppression hybridization of the product
to
Cot
1 or genomic DNA to remove repeat
sequences before the product is used in
the screening of recombinant libraries.
4.2
Analysis of Cloned DNA
Once a chromosomal region–speciFc re-
combinant DNA library has been con-
structed, it should be tested for its com-
pleteness and complexity. One should also
verify whether the cloned DNA originated
from the microdissected fragment of inter-
est. ±irst, DNA insert size and range must
be determined. Second, the percentage of
clones containing repeat and unique se-
quences must be calculated. Third, the
percentage of the total microdissected
DNA fragments that has been cloned is
determined. ±ourth, cloned sequences are
localized to the respective chromosomal re-
gions by
in situ
hybridization or Southern
hybridization to region-speciFc DNA.
±or determination of the average DNA
insert size and range, a number of clones
(30–50) are picked at random, and the
insert DNA is released from the cloning
vector by digestion with the appropriate
restriction enzyme and is fractionated on
an agarose or polyacrylamide gel elec-
trophoresis apparatus using appropriate
DNA markers. The restriction enzyme di-
g
e
s
t
iono
fe
a
cho
fth
er
andom
l
yp
i
ck
ed
clones provides a pattern that is indicative
o
fthenumbero
fun
iquec
lonesinthel
i
-
brary. Estimation of the average size and
range of cloned inserts as well as deter-
mination of the number of independent
clones is used to calculate the complete-
ness of the library.
Next, the random clones can be used to
identify the portion of recombinant clones
that contain repeat sequences. DNA in-
serts are transferred to a nitrocellulose
Flter and probed with total genomic hu-
man DNA that is radioisotopically labeled
by nick translation. If the library were gen-
erated in a completely unbiased manner,
al
a
rg
enumb
e
r(
80%) of these clones
would contain repeat sequences and would
result in positive hybridization. Unique
sequences are determined by digesting to-
tal genomic DNA with several restriction
enzymes (e.g.
Bam
HI,
Eco
RI,
Pst
I) and
performing a Southern hybridization. Re-
combinant DNA inserts from individual
clones are radioisotopically labeled and
used to hybridize to restriction polymor-
phic genomic fragments under conditions
that favor unique sequences (65–68
C).
An ideal library generated from a chro-
mosome fragment would consist of a
series of recombinant DNA clones con-
taining overlapping sequences that span
the entire chromosomal region. The size
of a library of completely random frag-
ments of genomic DNA needed to ensure
representation of a particular sequence of
interest is dictated by the size and num-
ber of the cloned inserts and the size of
the chromosomal fragment originally dis-
sected. The following formulas are used
to calculate the percentage of DNA that
would be cloned from a chromosome frag-
ment. Theoretically, the number of clones
that is required to achieve a library of a
99% probability of containing a particular
sequence is
N
=
ln
(
1
P
)
ln
(
1
I
/
G
)
where
P
is the desired probability,
I
is
the insert size,
G
i
st
h
eg
e
n
om
es
i
z
e
of the chromosome fragment, and
N
is the necessary number of clones. ±or
example, to generate a recombinant library
of average insert size 2.4 kb (a typical
insert size following
Eco
RI digestion), the
number of clones necessary to ensure
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