A rapid and non‐radioactive PCR based assay for the detection of allelic loss in human gliomas

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Abstract
Studies of the loss of allelic heterozygosity (LOH) in tumour tissues have evolved as an important tool for the identification of chromosomal regions which are likely to harbour tumour suppressor genes. The classical procedure to determine LOH has been restriction fragment length polymorphism (RFLP) analysis and Southern blotting, a time consuming method requiring radioisotopes and several micrograms of DNA. Recently, the use of highly polymorphic microsatellites of the CA-dinucleotide repeat class and polymerase chain reaction (PCR) has considerably advanced and facilitated the detection of LOH in tumour tissues. We here describe a strategy to identify LOH based on PCR amplification of CA-dinucleotide repeats, denaturing polyacrylamide gel electrophoresis (PAGE) and nucleic acid detection with a sensitive silver staining protocol. In a comparative study of 20 astro-cytomas, this rapid technique was able to identify all cases of LOH on chromosome 17 p that had previously been found in these tumours by RFLP analysis and Southern blotting. This non-radioactive PCR based assay has a great potential for LOH studies in human tumours.