AMPLIFICATION AND DETECTION OF A Y-CHROMOSOME DNA-SEQUENCE BY FLUORESCENCE IN-SITU POLYMERASE CHAIN-REACTION AND FLOW-CYTOMETRY USING CELLSIN SUSPENSION

A procedure for amplifying and detecting nucleic acid sequences in sit u using cells in suspension and flow cytometry has been developed, The process involves the use of the polymerase chain reaction (PCR) and a fluorescent in situ hyhridization (FISH) protocol developed in our la boratory to detect the amplified PCR product, For these studies, a Y-c hromosome specific repeat DNA sequence was amplified, Daudi cells, a B -cell lymphoma culture line established from a male, was used as a pos itive control and HL-60, a promyelocytic leukemia culture line establi shed from a female, was used as a negative control, During the in situ PCR process cellular autofluorescence (noise) increases causing marke dly reduced detection sensitivity of the probe (signal) bound to the a mplified product within the positive cells, An autofluorescence reduct ion circuit was applied which was integrated into a standard bench top flow cytometer to reduce this noise, thereby producing a IO-fold incr ease in detection sensitivity of the signal, Without the application o f the autofluorescence reduction circuit, the positive control histogr am distribution was virtually indistinguishable from the negative cont rol sample distributions, After autofluorescence reduction, the data s howed that the Y-chromosome DNA was only amplified in the Daudi cells subjected to the complete in situ PCR protocol, This increased sensiti vity also provided direct detection of the Y-chromosome repeat sequenc e, albeit exhibiting less signal compared to the amplified target afte r the in situ PCR. (C) 1995 Wiley-Liss, Inc.