Manual 768 or 384 well microplate gel 'dry' electrophoresis for PCR checking and SNP genotyping

Abstract

Electrophoresis continues to be a mainstay in molecular genetic laboratories for checking, sizing and separating both PCR products, nucleic acids derived from in vivo or in vitro sources and nucleic acid-protein complexes. Many genomic and genetic applications demand high throughput, such as the checking of amplification products from many loci, from many clones, from many cell lines or from many individuals at once. These applications include microarray resource development and expression analysis, genome mapping, library and DNA bank screening, mutagenesis experiments and single nucleotide polymorphism (SNP) genotyping. PCR hardware compatible with industry standard 96 and 384 well microplates is commonplace. We have previously described a simple system for submerged horizontal 96 and 192 well polyacrylamide or agarose microplate array diagonal gel electrophoresis (MADGE) which is microplate compatible and suitable for PCR checking, SNP typing (restriction fragment length polymorphism or amplification refractory mutation system), microsatellite sizing and identification of unknown mutations. By substantial redesign of format and operations, we have derived an efficient 'dry' gel system that enables direct 96 pin manual transfer from PCR or other reactions in microplates, into 768 or 384 well gels. Combined with direct electrode contact in clamshell electrophoresis boxes which plug directly to contacts in a powered stacking frame and using 5-10 min electrophoresis times, it would be possible (given a sufficient supply of PCRs for examination) for 1 million gel tracks to be run per day for a minimal hardware investment and at minimal reagent costs. Applications of this system for PCR checking and SNP genotyping are illustrated.

Mutation scanning by meltMADGE: Validations usingBRCA1andLDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population

“…MADGE formers and glass plates were as previously described (Day and Humphries 1994;Gaunt et al 2003). Large batches of gels were poured in a purpose-built pouring box (Supplemental Fig.…”

“…Electrophoresis was for 2-3 h at 50 V and 2 A with a linear ramp temperature from 59°C to 64°C (LDLR exon 3) or from 60°C to 65°C (LDLR exon 8), regularly calibrated to national standards. Gels were stacked separated by spacers for staining in 100 mL of ‫ן1‬ TAE buffer with 10 µL of Vistra Green (Molecular Probes) on a shaker at minimum speed for 15 min, and visualized using a Fluorimager 595 (Molecular Dynamics, Amersham Pharmacia Biotech) as described (Gaunt et al 2003). Ethidium bromide and a UV transilluminator can also be used.…”

“…1 and Methods) for population studies. Melt-MADGE combines the properties of MADGE (Day and Humphries 1994;Gaunt et al 2003) with a reconfiguration of denaturing gradient gel electrophoresis (DGGE) (Fischer and Lerman 1979), using a thermal ramp in time rather than a linear gradient in space, to increase the sample parallelism and reduce the costs of mutation scanning by one to two orders of magnitude. Here we describe the development of the method, its validation in the detection of unknown single base and small insertion/deletion variants in BRCA1 and LDLR, and the study of regions of the LDLR gene in relation to cholesterol levels in population samples representing ∼8000 subjects.…”

Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting

“…For this purpose the technique known as microplate array diagonal gel electrophoresis (MADGE) has been developed. MADGE is an electrophoresis system with a large number of wells placed askew in a separating matrix of polyacrylamide or agarose (Gaunt et al, 2003). The number of wells can be as large as 384 or 768 permitting rapid transfer of samples from microplates.…”

Madge - Microplate Array Diagonal Gel Electrophoresis

“…Day et al (6) reconfigured the MADGE format and associated procedures and made a 768 well-MADGE. A design was adopted which uses an 11.3°a ngle between the 12 well rows of each intercalated 96 well component array and the direction of electrophoresis.…”