A new technique for cyclic in situ amplification and a case report about amplification of a single copy gene sequence in human metaphase chromosome through PCR-PRINS

Since the introduction of PRimed IN Situ labeling (PRINS) as a rapid and ex tremely sensitive alternative method to conventional fluorescence in situ h ybridization (FISH), its application in clinical cytogenetics has been limi ted to the detection of highly repeated sequences, such as centromeric and telomeric regions. In the original PRINS method, unlabeled oligonucleotide probes are annealed to their repeated complementary target sequences in fix ed human metaphase chromosomes on a slide. The probes serve as primers for subsequent in situ chain elongation with Tag DNA polymerase and labeled nuc leotides. In contrast to conventional PCR, cyclic in situ amplification of the chromosomal target DNA with paired primers remained both difficult and strictly limited to highly repeated sequences, since the maintenance of con stant reaction conditions on the slide during temperature and pressure shif ts presents a major problem. We developed a new system for in situ PCR that allows the amplification of target sequences analogous to PCR in the test tube, We applied this method successfully for the detection of highly repea ted sequences, for the detection of low copy repeats, and in one case, for the detection of a single-copy DNA sequence. The significance of this devel opment for further in situ PCR applications will be discussed. Hum Mutat 17 :131-140, 2001, (C) 2001 Wiley Liss, Inc.