Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations
Inm. Day et al., Microplate array diagonal gel electrophoresis (MADGE), CpG-PCR and temporal thermal ramp-MADGE (Melt-MADGE) for single nucleotide analyses in populations, GENET A-BIO, 14(5-6), 1999, pp. 197-204
Important requirements for molecular genetic epidemiological studies are ec
onomy, sample parallelism, convenience of setup and accessibility, goals in
adequately met by existent approaches. We invented microplate array diagona
l gel electrophoresis (MADGE) to gain simultaneously the advantages of simp
le setup, 96-well microplate compatibility, horizontal electrophoresis, and
the resolution of polyacrylamide. At essentially no equipment cost (one si
mple plastic gel former), 10-100-fold savings on time for sample coding, li
quid transfers, and data documentation, in addition to volume reductions an
d gel re-use, can be achieved. MADGE is compatible with ARMS, restriction a
nalysis and other pattern analyses. CpG-PCR is a general PCR approach to Cp
G sites (10-20% of all human single base variation): both primers have 3' T
, and are abutted to the CpG, forcing a TaqI restriction site if the CpG is
intact. Typically, a 52 bp PCR product is then cut in half. CpG-PCR also i
llustrates that PAGE-MADGE readily permits analysis of 'ultrashort' PCRs. M
elt-MADGE employs real-time-variable-temperature electrophoresis to examine
duplex mobility during melting, achieving DGGE-like de novo mutation scann
ing, but with the conveniences of arbitrary programmability, MADGE compatib
ility and short run time. This suite of methods enhances our capability to
type or scan thousands of samples simultaneously, by 10-100-fold. (C) 1999
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