AMPLIFICATION AND DETECTION OF A Y-CHROMOSOME DNA-SEQUENCE BY FLUORESCENCE IN-SITU POLYMERASE CHAIN-REACTION AND FLOW-CYTOMETRY USING CELLSIN SUSPENSION
Ea. Timm et al., AMPLIFICATION AND DETECTION OF A Y-CHROMOSOME DNA-SEQUENCE BY FLUORESCENCE IN-SITU POLYMERASE CHAIN-REACTION AND FLOW-CYTOMETRY USING CELLSIN SUSPENSION, Cytometry, 22(3), 1995, pp. 250-255
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.