M
olecular-genetic epidemiological studies demand
economy, the ability to assay many samples
simultaneously, convenient setup and accessi-
bility for both small and large laboratories. These
objectives are generally inadequately met by existing
approaches. Systematic studies of associations between
genotypic variation and disease traits will demand the
analysis of many thousands of candidate variations,
commonly single-nucleotide variations. Power studies
1
indicate that, for common disease traits to which indi-
vidual genotypes make small contributions, studies
comprising hundreds to thousands of nuclear families
or individuals will be essential. Multipoint analysis of
panels of single-nucleotide polymorphisms may, in
the future, take over from microsatellite-based genome
scans for linkage studies
2
. Such studies can be prohibi-
tively expensive and one of the major hurdles to
overcome is thus cost reduction.
Our philosophy has been that electrophoresis still has
many advantages over homogeneous systems, such as
analysis of size, shape and charge of molecules. Elec-
trophoresis is also well known and available to all users,
requires no expensive hardware (implicit in homo-
geneous techniques) and has a range of well-established
methodologies [allele-specific amplification, restric-
tion-fragment-length-polymorphism (RFLP) analysis,
heteroduplex analysis, denaturing gradient-gel elec-
trophoresis (DGGE), single-strand-conformation-
polymorphism (SSCP), protein electrophoresis etc.].
However, gel preparation, long tracks (requiring more
cumbersome equipment and longer run times), incom-
patibility with industry-standard microplates and ver-
tical-format polyacrylamide gels (restricting the num-
ber of analyses per gel because only one row of samples
can be loaded at the top of the gel) make electrophoresis
an unattractive (although much-used) option to under-
pin laboratory studies of genetic diversity within popu-
lations. We have explored short-track electrophoresis
and approaches to electrophoresis compatible with
the requirements of molecular-genetic epidemiology
outlined above
3–10
.
Microplate-array diagonal-gel electrophoresis
Electrophoresis of DNA is traditionally performed in
either an agarose- or a polyacrylamide-gel matrix.
Much effort has been directed to improved-quality
agaroses capable of higher resolution but, for small
fragments such as those from PCR and post-PCR
digests, polyacrylamide still offers the highest resolu-
tion. Although agarose gels can easily be prepared in an
open-faced format [uncovered, with well-forming
comb(s) inserted in the molten substrate] to gain the
convenience of horizontal electrophoresis, acrylamide
does not polymerize in the presence of air and the usual
configuration is vertical between two glass plates. In this
article, we describe a very simple device and method
to prepare and manipulate horizontal polyacrylamide
gels. In addition, the open-faced horizontal arrange-
ment enables arrays of wells to be loaded. As many pro-
cedures are undertaken in standard 96-well microtitre
plates, we have also designed a device that preserves the
exact configuration of the 8 ϫ 12 array.
The original MADGE format
3,4
requires at least a
two-piece kit (MadgeBio, Grantham, UK) consisting
of a two-dimensional plastic former and a single glass
plate coated with γ-methacryloxypropyltrimethoxy-
silane. The plastic former contains a 2-mm-deep,
100 ϫ 150 mm rectangular ‘swimming pool’ within
which (in our preferred format) there are 96 2-mm
cubic ‘teeth’ (well formers) in an 8 ϫ 12 array (with a
9 mm pitch) directly compatible with 96-well plates.
The array is set on an angle of 71.6Њ to the long side of
the ‘pool’, which is parallel to the direction of elec-
trophoresis, giving tracks that can pass between two
succeeding rows of wells – an angle of 71.6Њ gives 26.5-
mm tracks bounded cathodally by the sample well for
that track and anodally by another well (Fig. 1). With
a larger angle, longer tracks can be achieved at the
TIBTECH JULY 1998 (VOL 16)
Copyright ©1998, Elsevier Science Ltd. All rights reserved. 0167 - 7799/98/$19.00. PII: S0167-7799(98)01217-7
287
F
OCUS
Microplate-array diagonal-gel electrophoresis
(MADGE) and melt-MADGE: tools for
molecular-genetic epidemiology
Ian N. M. Day, Emmanual Spanakis, Divya Palamand, Glenn P. Weavind and Sandra D. O’Dell
Microplate-array diagonal-gel electrophoresis (MADGE) was invented for molecular-genetic epidemiological studies. It com-
bines direct compatibility with microplates, convenient polyacrylamide-gel electrophoresis and economy of time and reagents
at minimal capital cost, and enables one user to run up to several-thousand gel lanes per day for the direct assay of single-
base variations. Melt-MADGE adds temporal-thermal-ramp apparatus to achieve similar throughput for
de novo
mutation
scanning.
I. N. M. Day (iday@hgmp.mrc.ac.uk), E. Spanakis, D. Palamand,
G. P. Weavind and S. D. O’Dell are at the Wessex Human Genet-
ics Institute, University of Southampton, Duthie Building (Mp 808),
Southampton University Hospitals NHS Trust, Tremona Road,
Southampton, UK SO16 6YD.