ABSTRACT
We have developed a vectorette PCR approach to provide an improved method for
isolation of microsatellite repeats. The modified procedure relies on PCR
amplification using a vectorette-specific primer in combination with one of a panel of anchored
dinucleotide repeat primers The target DNA to be screened for microsatellite
sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have
used this technique to isolate novel, polymorphic microsatellite repeats from
clones containing the amelogenin gene (AMGX) located on human chromosome
Xp22.3.
Despite the large numbers of human microsatellite markers now available, the
identification and isolation of additional, novel, highly polymorphic DNA
markers remains important. Such markers are needed for the refinement of
genetic linkage maps, gene-disease association studies, pre-natal diagnosis using linked markers and loss of heterozygosity
analyses of paired normal and tumour DNA samples. Polymorphic markers are also
required for the construction of genetic maps for economically important animal
and plant species. At present, identification and isolation of polymorphic microsatellite sequences involves screening of cloned DNA fragments with radioactively labelled di-, tri- or tetra-nucleotide repeat oligonucleotide probes. Positively
hybridising clones are then isolated and DNA extracted and purified for
sequence analysis. Oligonucleotide primers flanking the microsatellite sequence
are designed and used for amplification of the repeat. This approach is time-consuming, particularly when large numbers of clones have to be plated,
screened, isolated and sequenced.
We have developed a vectorette PCR approach to provide an improved method for
isolation of microsatellite repeats. Vectorette PCR is a linker-specific amplification technique originally used for the isolation of
terminus-specific sequences from large cloned DNA inserts (
1
). The modifed procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of 12 anchored
dinucleotide repeat primers: d(AC)
10
C, d(AC)
10
G, d(AC)
10
T, d(CA)
10
A, d(CA)
10
G, d(CA)
10
T, d(GT)
10
A, d(GT)
10
C, d(GT)
10
T, d(TG)
10
A, d(TG)
10
C and d(TG)
10
G. The target DNA to be screened for microsatellite sequences can be from YAC,
Pl, cosmid, bacteriophage or plasmid clones. An outline of the technique is
presented in Figure
1
.
This approach generates DNA sequence from one side flanking the microsatellite
repeat. A reverse primer is designed from this sequence, which in combination
with the universal vectorette primer, is used to re-amplily the vectorette libraries. The reverse primer will prime back
through the repeat and permit sequencing of flanking DNA on the other side of
the microsatellite repeat. This will then allow a pair of primers flanking the
microsatellite to be generated for routine PCR analysis. We tested the ability
of this technique to isolate novel, polymorphic dinuclcotide repeat sequences
from vectorette libraries made from plasmid, cosmid and yeast artificial
chromosome (YAC) clones. The clones used in the study contained part (plasmid)
or all (cosmid and YAC clones) of the amelogenin gene (AMGX) sequence, which
maps to human chromosome Xp22.3, as determined by PCR analysis using exon
specific primers (
2
).
Six separate vectorette libraries were constructed using 50 ng- 1 [mu]g cloned DNA (depending on target DNA complexity) restriction enzyme
digested for 1 h/37oC in 50 [mu]l 1* reaction buffer containing 20 U of each of one of the following
enzymes:
Alu
I,
Bgl
II,
Dpn
II,
Eco
RV,
Rsa
I or
Sau
3A. A range of different restriction enzymes was used so as to increase the
chance that the restriction fragment containing the microsatellite repeat was
of an amplifiable size. Three picomoles (5 [mu]l) of the appropriate vectorette unit (
1
) were ligated to the restriction fragments in a total volume of 10 [mu]l containing 1 [mu]l 100 mM ATP, 1 [mu]l 100 mM DTT and 1 U T4 DNA ligase. Ligation reactions were
incubated at 20oC for 60 min followed by 37oC for 30 min. The whole process was repeated three times. This is
necessary to re-digest any target DNA fragments which have ligated to each other and not
to vectorette units. If a 4 bp cutting enzyme is used whose site is reformed on
ligation (e.g.
Sau
3A) the restriction digest must be heat denatured (70oC/10 min) before ligation. Following ligation, 100 [mu]l water was added to the vectorette library. Since yeast genomic DNA
also contains microsatellite repeats that will co-amplify with human DNA during PCR, it is preferable to isolate the
individual YAC band from a low melting point agarose pulsed-field gel prior to vectorette library construction. Following pulsed-field gel electrophoresis, the YAC band (~430 kb) containing the amelogenin gene was identified by
ethidium bromide staining and excised from the gel. The agarose gel slice
containing the YAC band was placed in a 1.5 ml microfuge tube and equilibrated
overnight in 1* restriction enzyme buffer. The buffer was removed and the agarose gel
slice melted by heating at 65oC. DNA (30 [mu]l) was used for restriction enzyme digestion in a 50 [mu]l volume as described above.
REFERENCES
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