Genomic analysis of human multigene families using chromosome-specific vectorette PCR
Genomic analysis of human multigene families using chromosome-specific vectorette PCR
Terry P.
Moynihan
1,
*
,
Alexander F.
Markham
1
and
Philip A.
Robinson
1,2
1
Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St
James's University Hospital,
Leeds
LS9 7TF,
UK
and
2
Leeds Dental Institute, University of Leeds,
Leeds
LS2 9LU,
UK
Received July 21, 1996;
Revised and Accepted August 9, 1996
ABSTRACT
We report a technique for the rapid determination of genomic structure of
individual members of human interspersed multigene families which circumvents
the requirement for genomic clone isolation. In this approach, vectorette
libraries were constructed from human/rodent somatic cell hybrid DNA harbouring
single members of the gene family. Using these libraries as PCR templates with
nested gene-specific primers in combination with a common vectorette primer resulted
in the amplification of gene-specific products suitable for the subsequent determination of intron/exon
structure. We have applied this technique to characterise members of two gene
families.
Vectorette PCR is a technique for the elucidation of unknown DNA sequence
adjacent to a region of known sequence. Initially reported for the
characterisation of genomic sequence at YAC insert/vector junctions (
1
), it has subsequently been used to determine gene structure using vectorette
libraries constructed from bacteriophage and YAC genomic clones (
2
,
3
). Other workers have circumvented the time-consuming step of first isolating genomic clones through the use of
libraries constructed from total genomic DNA (
4
-
6
). However, this approach is limited since many genes are members of multigene
families that share considerable nucleic acid sequence homology. As the
specificity of vectorette PCR is reliant upon the use of a sequence- specific primer in combination with a common vectorette primer (
1
), the use of a gene-specific primer derived from the known sequence of one gene may result in
the co-amplification of sequence from other family members thus confounding their
characterisation. We have resolved this problem, without clone isolation, by
constructing vectorette libraries using DNA derived from appropriate somatic
cell hybrids containing single human chromosomes harbouring the genes of
interest.
We first tested the capacity of this technique to amplify sequence from exon
8/intron 8 of the presenilin 1 (
S182
) gene, the gene for early-onset familial Alzheimer's disease, localised on chromosome (Chr) 14q24.3
(
7
). This gene is a member of a multigene family of which at least one other
member, encoding presenilin 2
(STM2),
has been identified on Chr 1q31-42 (
8
). The efficacy of the technique was assessed using as templates vectorette
libraries constructed from human genomic DNA, mouse genomic DNA, YAC clone
(30FF7) DNA containing exons 7-12 of the
S182
gene, and DNA isolated from the the human monochromosomal cell lines (GM10479
and GM13139, Coriell Cell Repositories) which contain human Chr 14 and Chr 1
respectively on a mouse DNA background. DNA (1 [mu]g) was digested with 20 U each of the four base-pair recognition site restriction enzymes
Alu
I,
Hae
III,
Rsa
I and
Sau
3AI in a final volume of 20 [mu]l at 37oC for 2 h. The reactions were terminated by heat-inactivation at 85oC for 30 min. Equimolar amounts of the phosphorylated blunt-end or
Bam
HI vectorette unit (
1
) were ligated to the restriction digested DNA, in a final reaction volume of 25
[mu]l containing 0.5 mM ATP, 10 mM DTT and 1.5 U T4 DNA ligase (Promega) at 20oC for 2 h. The ligase was then heat- inactivated by incubation at 70oC for 20 min and water was added to the vectorette libraries
to a final volume of 100 [mu]l, thus generating four libraries for each of the DNA samples under
analysis. Five microlitres of each vectorette library was used as a PCR
template with the vectorette primer (5'-dCGAATCGTAACCGTTCGT) and an
S182
-specific primer (5'-dATTTAGTGGCTGTTTTGTG) in a final reaction volume of 50 [mu]l containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl
2
, 200 [mu]M dNTPs and 0.2 [mu]M each primer. `Hot-start' PCR was performed (
9
), by initially denaturing the template DNA at 95oC for 5 min and then lowering the temperature to 90oC before the addition of 2 U of
Taq
polymerase (Promega). Thirty five cycles of 95oC for 30 s, 55oC for 30 s and 72oC for 90 s were then carried out, followed by a final 5 min
extension step at 72oC. After the first round of PCR, products generated from the somatic cell
hybrid cell lines and genomic DNA vectorette libraries were visualised as a
faint smear by agarose gel electrophoresis, a common feature of this technique
when using complex DNA templates. A second round of hemi-nested PCR was then performed, using a 5000-fold dilution of the primary PCR reaction. When an internal gene- specific primer (5'-dTTGAAACAGCTCAGGAGAGA) was used in conjunction
with the vectorette primer a distinct PCR product was observed. In contrast,
when hemi-nested PCR was performed nesting the vectorette primer (5'-dCGAATCGTAACCGTTCGTACGAGAATCGCT) rather than the gene-specific primer, a smeared gel profile similar to that
obtained in the primary PCR resulted. It was only necessary to nest the gene-specific primer; no improvement was observed by nesting both the gene-specific and vectorette primers.
A PCR product of ~800 bp was amplified from the
Hae
III vectorette libraries generated from human genomic DNA, YAC clone 30FF7, and
the human Chr 14 somatic cell hybrid cell line GM10479 (Fig.
1
A). It was absent in the negative control libraries derived from yeast DNA,
mouse DNA and another monochromosomal somatic cell hybrid (GM13139) containing
human Chr 1 DNA which harbours the
STM2
gene. The PCR products were gel- purified, and directly sequenced using the
fmol
[
DNA sequencing system (Promega) with both a [gamma]-
32
P end-labelled nested gene- specific primer and also a nested vectorette sequencing primer (5'-dAGAATCGCTGTCCTCTCCTT). Sequence comparison revealed that the same product had been amplified from
YAC clone, monochromosomal cell line and human genomic DNA derived libraries,
and that the sequence obtained corresponded to published data (
7
,
10
).
2 Dyer,K.D. and Rosenberg,H.F. (1995) Biotechniques19,550-552.
3 Roberts,R.G., Coffey,A.J., Bobrow,M. and Bentley,D.R. (1992) Genomics13,942-950.
4 Arnold,C. and Hodgson,I.J. (1991) PCR Methods Appl.1, 39-42.MEDLINE Abstract
5 Will,K., Dörk,T., Stuhrmann,M., Meitinger,T., Bertele-Harms,R., Tümmler,B. and Schmidtke,J. (1994) J. Clin. Invest.93, 1852-1859.MEDLINE Abstract
6 Schoenmakers,E.F.P.M., Mols,R., Wanschura,S., Kools,P.J.F., Geurts,J.M.W., Bartnitzke,S., Bullerdiek.,J., Van den Berghe,H. and Van de Ven,W.J.M. (1994) Genes Chromosom. Cancer11, 106-118.
