Microplate-array diagonal-gel electrophoresis (MADGE) and melt-MADGE: tools for molecular-genetic epidemiology

Day, Ian N.M.; Spanakis, Emmanuel; Palamand, Divya; Weavind, G.P.; O’Dell, Sandra D.

doi:10.1016/s0167-7799(98)01217-7

Cited by 20 publications

(7 citation statements)

References 13 publications

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“…The primary long PCR reaction was checked using both ethidium bromide staining of agarose gels and also using the 10-fold more sensitive approach of Vistra Green4 staining and fluorimaging (Molecular Dynamics, Sunnyvale, CA). The nested amplification of CYP2A6 exon 3 and its subsequent digestion by XcmI and DdeI for positive identification of two variant alleles was essentially as described by Fernandez-Sanguero et al All PCR reactions and digests were examined by electrophoresis in MADGE (microplate array diagonal gel electrophoresis) gels (Day et al 1998), as shown in Figure 1.…”

Section: Cyp2a6 Genotypingmentioning

confidence: 99%

The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit

Hinks²,

Morton³

et al. 2000

Annals of Human Genetics

115

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Long PCR followed by nested PCR has previously been used to determine CYP2A6 160H alleles, but the method proved unreliable. We have optimized this approach in a DNA bank of 1032 subjects (age range 59-74 years) to give reliable results, yielding indirect molecular evidence and very strong statistical evidence of hitherto unrecognized common alleles (designated O) recalcitrant to the long PCR. Coding three alleles (160L, 160H and O) and an approach to association analysis originally developed to deal with null alleles implicit in ABO blood group phenotyping, the contribution of 160H (functionally null) to reduced smoking habit has been clearly measured for the first time, unconfounded by alleles null to the long PCR. The most significant findings (p 0n01) are that the possession of a 160H allele, compared with not possessing a 160H allele, is associated with a mean age of starting regular smoking 3 years later (95 % CIp1n93 years, average start age 20-21 years rather than 17-18 years) ; and that the average likelihood of quitting smoking at any time is 1n75 fold (95 % CI. 1n17-2n61) for those possessing an 160H allele compared with those who have no 160H allele. This suggests that a smoking subject with a genotype predicted to confer 50 % of the ability to eliminate nicotine via the CYP2A6 pathway has almost twice the likelihood of quitting smoking. CYP2A6 belongs to the CYP2 family of P450 cytochromes responsible for the metabolism of xenobiotics including nicotine. A 350 kb contig on human chromosome 19 containing CYP2A6 and related genes and pseudogenes was characterised by Fernandez-Salguero et al. (1995). The 160H allele of CYP2A6 is unable to metabolise coumarin and has low nicotine to cotinine conversion (Yamano et al. 1990 ;Oscarson et al. 1998). Reduced risk of tobacco dependence and reduced total consumption have been reported

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Section: Cyp2a6 Genotypingmentioning

confidence: 99%

The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit

Hinks²,

Morton³

et al. 2000

Annals of Human Genetics

115

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“…Day et al (8,9) described temporal-thermalramp electrophoresis MADGE (melt MADGE), in which the analysis of duplex melting provides a method of de novo mutation scanning. Using a purpose-built temporal-thermal-ramp-electrophoresis apparatus for programmable melting display (PMD) (melt-MADGE), a DGGE-like analysis was undertaken in MADGE format.…”

Section: Temporal Thermal Ramp Electrophoresis Madge (Melt-madge)mentioning

confidence: 99%

Madge - Microplate Array Diagonal Gel Electrophoresis

Stanković¹,

Alavantić²,

Majkić-Singh³

2009

Journal of Medical Biochemistry

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Madge - Microplate Array Diagonal Gel ElectrophoresisMicroplate array diagonal gel electrophoresis (MADGE) was invented for molecular genetic epidemio-logical studies. MADGE is a highly flexible, cost effective, microplate compatible solution to high throughput electrophoresis needs. It enables several thousands to million gel lines per day for direct assay of single-base variation in different capacity laboratories. Variants of the standard 96-well MADGE include: 96-well stretch-MADGE, 192-well MADGE, 384-well MADGE, and 768-well MADGE. Melt-MADGE combines the temporal thermal ramp apparatus to achieve similar throughput for de novo mutation scanning. Basic MADGE principles and procedures, preparation of MADGE gels, electrophoresis, visualization and analysis of these gels, as well as modifications of the basic 96-well MADGE will be discussed in detail. For the first time in our country, this revolutionary polyacrylamid electrophoresis was done in 1998. We shortly review our studies which used MADGE for high throughput genotyping of the apolipoprotein E. MADGE and melt-MADGE will have an important role in the future genetic research of complex diseases and especially in pharmacogenomics.

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“…DNA was extracted using a standard protocol and analysed for the presence or absence of the two mutations using standard techniques [19][20][21]. Homozygosity for the C282Y mutation (HHYY) and compound heterozygosity for both the C282Y mutation and the H63D mutation (HDCY) were categorised as 'positive' screening results.…”

Section: Genotypicmentioning

confidence: 99%

Factors affecting the uptake of screening: A randomised controlled non-inferiority trial comparing a genotypic and a phenotypic strategy for screening for haemochromatosis

Patch

Roderick

Rosenberg

2005

Journal of Hepatology

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Microplate-array diagonal-gel electrophoresis (MADGE) and melt-MADGE: tools for molecular-genetic epidemiology

Cited by 20 publications

References 13 publications

The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit

The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit

Madge - Microplate Array Diagonal Gel Electrophoresis

Factors affecting the uptake of screening: A randomised controlled non-inferiority trial comparing a genotypic and a phenotypic strategy for screening for haemochromatosis

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