A new technique for cyclic in situ amplification and a case report about amplification of a single copy gene sequence in human metaphase chromosomes through PCR-PRINS.
Since the introduction of PRimed IN Situ labeling (PRINS) as a rapid and extremely sensitive alternative method to conventional fluorescence in situ hybridization (FISH), its application in clinical cytogenetics has been limited 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 fixed human metaphase chromosomes on a slide. The probes serve as primers for subsequent in situ chain elongation with Taq DNA polymerase and labeled nucleotides. 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 constant reaction conditions on the slide during temperature and pressure shifts 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 repeated 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 development for further in situ PCR applications will be discussed.
Harrer
T
T
Department of Human Genetics, Medical University of Lübeck, Lübeck, Germany. mennicke@medinf.mu-luebeck.de
Schwinger
E
E
Mennicke
K
K
D001054:Apolipoproteins A / Q000235:genetics; D002877:Chromosomes, Human / Q000235:genetics*; D017931:DNA Primers; D016189:Dystrophin / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D006801:Humans; D008297:Male; D008677:Metaphase / Q000235:genetics*; D009136:Muscular Dystrophies / Q000235:genetics; D016133:Polymerase Chain Reaction; D020401:Primed In Situ Labeling / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics
D001054:Apolipoproteins A; D017931:DNA Primers; D016189:Dystrophin