Biomedical Engineering Reference
In-Depth Information
Box 6.4 A landmark publication. Identification of the cystic
fibrosis gene by chromosome walking and jumping
Cystic fibrosis (CF) is a relatively common severe
autosomal recessive disorder. Until the CF gene was
cloned, there was little definite information about the
primary genetic defect. The cloning of the CF gene
was a breakthrough for studying the biochemistry of
the disorder (abnormal chloride-channel function),
for providing probes for prenatal diagnosis and for
potential treatment by somatic gene therapy or other
means. The publication is especially notable for the
generality of the cloning strategy. In the absence of
any direct functional information about the CF gene,
the chromosomal location of the gene was used as
the basis of the cloning strategy. Starting from
markers identified by linkage analysis as being close
to the CF locus on chromosome 7, a total of about
500 kb was encompassed by a combination of
chromosome walking and jumping. Jumping was
found to be very important to overcome problems
caused by 'unclonable' regions which halted the
sequential walks, and in one case achieved a distance
of 100 kb (Collins
et al
. 1987). In this work, large
numbers of clones were involved, obtained from
several different phage and cosmid genomic libraries.
Among these libraries, one was prepared using the
Maniatis strategy using the
l
Charon 4A vector, and
several were prepared using the
l
DASH and
l
FIX
vectors (Fig. 6.4) after partial digestion of human
genomic DNA with
Sau
3AI. Cloned regions were
aligned with a map of the genome in the CF region,
obtained by long-range restriction mapping using
rare-cutting enzymes, such as
Not
I, in combination
with pulsed-field gel electrophoresis (p. 10). The
actual CF gene was detected in this cloned region
by a number of criteria, such as the identification
of open reading frames, the detection of cDNAs
hybridizing to the genomic clones, the detection
of cross-hybridizing sequences in other species and
the presence of CpG islands, which are known
to be associated with the 5
′
ends of many genes
in mammals.
From: Rommens
et al.
(1989)
Science
245: 1059-65.
generated by digestion with endonucleases, such
as
Not
I, which cut at very rare target sites. This
is followed by subcloning of the region covering
the closure of the fragment, thus bringing together
sequences that were located a considerable distance
apart. In this way a
jumping library
is constructed,
which can be used for long-distance chromosome
walks (Collins
et al
. 1987, Richards
et al
. 1988). The
application of chromosome walking and jumping to
the cloning of the human cystic fibrosis gene is dis-
cussed in Box 6.4.
but only if there is sufficient information about its
sequence to make suitable primers.*
To isolate a specific clone, PCR is carried out with
gene-specific primers that flank a unique sequence
in the target. A typical strategy for library screening
by PCR is demonstrated by Takumi and Lodish
(1994). Instead of plating the library out on agar, as
would be necessary for screening by hybridization,
pools of clones are maintained in multiwell plates.
Each well is screened by PCR and positive wells are
identified. The clones in each positive well are then
* Note that, in certain situations, clever experimental design
can allow the PCR to be used to isolate specific but unknown
DNA sequences. One example of this is 5
Screening by PCR
The PCR is widely used to isolate specific DNA
sequences from uncloned genomic DNA or cDNA,
but it also a useful technique for library screening
(Takumi, 1997). As a screening method, PCR has
the same versatility as hybridization, and the same
limitations. It is possible to identify any clone by PCR
RACE, which is dis-
cussed on p. 101. Another is inverse PCR (p. 267), which can
be used to isolate unknown flanking DNA surrounding the
insertion site of an integrating vector. In each case, primers
are designed to bind to known sequences that are joined to the
DNA fragment of interest, e.g. synthetic homopolymer tails,
linkers or parts of the cloning vector.
′
