In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations |
| |
Authors: | L Paskins Jannine Brownie J Bull |
| |
Institution: | (1) Zeneca Diagnostics, Gadbrook Park, Northwich, Cheshire, CW9 7RA, UK, GB |
| |
Abstract: | 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.
Accepted: 15 February 1999 |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|