PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.
We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.
Chang
K S
KS
Department of Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Vyas
R C
RC
Deaven
L L
LL
Trujillo
J M
JM
Stass
S A
SA
Hittelman
W N
WN
D001483:Base Sequence; D002877:Chromosomes, Human / Q000737:chemistry*; D002886:Chromosomes, Human, Pair 17; D002891:Chromosomes, Human, Pair 21; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D005434:Flow Cytometry; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic
D015342:DNA Probes; D004273:DNA, Neoplasm; D004247:DNA