E-Book Overview
Cutting edge researchers demonstrate step-by-step how oligonucleotide primers may be successfully used to detect and amplify or extend complimentary sequences in situ. Through these procedures-often invented by the authors-the door is opened to rapid identification and characterization of chromosomal DNA sequences, viral genomes, and rare messenger RNAs in cells-at hitherto unmatched degrees of sensitivity and specificity. Their innovative techniques-suitable for both novice and experienced researchers-have rapidly become indispensable for many clinical diagnostic procedures, whether in quantification of chromosomes in the identification of aneuploidy for prenatal diagnosis, or in the identification of viral infection in the early stages, or of rare messenger RNAs present in cells.
E-Book Content
1 Oligonucleotide
PRINS DNA Synthesis
John R. Gosden and Diane Lawson 1. Introduction The technique for labeling chromosomesby annealing an oligonucleotide DNA primer to the denatured DNA of chromosome preparations on glass slides and extending it enzymatically in situ with the incorporation of labeled nucleotides was fust described by Koch et al. in 1989 (I). Since then, the technique has been greatly improved in reliability, sensitivity, and resolution, and now provides a viable, rapid alternative to conventional fluorescence in situ hybridization (FISH) for many investigations, particularly the identification of chromosome aneuploidy in metastatic tissues and antenatal diagnosis and the analysis of the human chromosome complement of somatic hybrid cell lines (Zd). 2. Materials 2.1. Primed In Situ Syf7thesis 1. Twin-Frost glassslides and 22 x 40 mm coverslips: The slides must be cleaned by soaking in ethanol to which a few drops of HCl have beenadded,followed by polishing with a cleanpiece of muslin, before the cells aredepositedon the slide. Coverslips must be