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PCR has revolutionised our ability to type for Single Nucleotide Polymorphisms (SNPs) but most detection methods to date have relied on the use of gels. Therefore, there is a requirement for a technique that would lend itself to automation and allow high throughput screening. AMDI (Alkaline Mediated Differential Interaction) is just such a system. It is a simple, inexpensive and effective way of detecting PCR product without the need for gels. AMDI makes use of the fluorescent properties of the dye SYBR-green 1. Under specialised conditions the dye binds to the PCR product, producing fluorescence detectable by fluorimeter. Previous attempts to use such dyes for this purpose have failed due to the high background fluorescence caused by reactants in the PCR. Using the commercially available buffer CABS (4-[cyclohexylamino]-1-butanesulphonic acid) the pH of the completed PCR can be increased at the time of reading, maintaining the fluorescence of the bound product while limiting that of the background. The difference in fluorescence between a positive and negative reaction is at least three fold. This system has been developed for HLA Class II, HLA Class I, Y chromosome and APC SNP typing. The HLA reactions have been validated using well characterised lymphoblastoid cell lines and we are now applying the AMDI system to the HLA typing of a number of populations. This widely applicable detection method is inexpensive, simple, robust and ultimately automatable. © 2001 Blackwell Science Ltd,.

Type

Journal article

Journal

European Journal of Immunogenetics

Publication Date

01/12/2001

Volume

28