PCR-SSCP-HDX analysis of pooled DNA for more rapid detection of germline
mutations in large genes. The BRCA1 example
PCR-SSCP-HDX analysis of pooled DNA for more rapid detection of germline mutations in large genes. The BRCA1 example
Piotr
Kozlowski
,
Krzysztof
Sobczak
,
Marek
Napierala
,
Marcin
Wozniak
,
Jakub
Czarny
and
Wlodzimierz J.
Krzyzosiak*
Laboratory of Cancer Genetics, Polish Academy of Sciences, Noskowskiego 12/14,
61-704
Poznan
,
Poland
Received September 12, 1995;
Revised and Accepted January 29, 1996
Several reports from laboratories involved in the isolation of the BRCA1 gene,
and recently in the search for its mutations (
1
-
5
), show that the latter activity is not a simple task. Every fragment of coding
sequence of this multi-exon gene has to be analysed as sequence variations may occur anywhere (
6
). Let us consider then some technical problems related to wide-scale screening of large genes for scattered germline mutations taking
BRCA1 as an example. Usually all BRCA1 exons, together with flanking intron
sequences or cDNA fragments, are sequenced directly. It is, however, labour
intensive. Alternatively, they are first screened by single strand conformation
polymorphism (SSCP) (
7
) to increase throughput of analysis. In the case of BRCA1 ~50 different gene fragments are analysed by SSCP and reamplified pure mutant
species are sequenced. It is also our experience (J. Czarny
et al.
, in preparation), that this approach becomes very laborious, if a large number
of chromosomes is to be tested. As no more reliable technology for large scale
mutation screening is at hand now, we took some measures to increase the
throughput of SSCP analysis without compromising its ability to detect
mutations. Assuming that the frequency of BRCA1 mutations is 0.5% in the
general population (
3
) screening of 200 individuals would give, on average, a single mutation.
Detecting this mutation would require running 200 SSCP gels (one gene fragment
from 50 individuals on one gel and 50 gene fragments analysed). The question we
addressed in this study was how to cut this number by a factor of 10 or more.
First, we have analysed the possibility of doing PCR-SSCP assay on genomic DNA pools of various size. The optimum number of
individual DNA samples to include in a pool is determined by the ability of
detecting variant SSCP bands by autoradiography. To test the limits of pooling,
the genomic DNA samples from different individuals were mixed together and
subjected to PCR followed by SSCP analysis which is in fact the SSCP-heteroduplex (HDX) analysis in the conditions we use. For these
experiments we have included the genomic DNA from patients in which mutations
or polymorphisms in BRCA1 were earlier found and carefully characterised (J.
Czarny
et al.,
in preparation). It turned out that in pools composed of four to five DNA
samples, fragments containing germline mutations could be easily detected.
In the case of germline mutations, one copy of the gene has a wild-type sequence and the other contains a mutation. According to our
experience this fraction of mutant DNA, when present in a variant SSCP band,
can be detected even when diluted 40 times with the signal from the wild-type sequence. However, in conditions of SSCP analysis, one or both DNA
strands very often form not a single but two or more unevenly represented
stable conformers showing different mobility. In the worst case, a new
conformer created by a mutation may contribute 5% or even less to the overall
radioactivity signal. To detect such less prominent signals, the number of DNA
samples combined in a pool should not be higher than four to five. Otherwise
they are barely discernible and could be missed. There are also other examples
where mutation does not change the gel migration of any of the single strand
conformers but the mobility of the heteroduplex formed is different. This is
sometimes observed in cases of very small insertion and deletion mutations. The
influence of DNA pooling on the ability of detecting these less prominent
variant bands is shown in Figure
1
.
