In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations.
Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling.
Paskins
L
L
Zeneca Diagnostics, Gadbrook Park, Northwich, Cheshire, UK. lynn.paskins@alderley.zeneca.com
Brownie
J
J
Bull
J
J
D001399:Azure Stains; D002460:Cell Line; D002891:Chromosomes, Human, Pair 21; D003116:Color; D000431:Ethanol; D005404:Fixatives; D018628:Gene Dosage; D006801:Humans; D008872:Microwaves; D020401:Primed In Situ Labeling / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation
D001399:Azure Stains; D005404:Fixatives; D000431:Ethanol