Isolation of 24 novel cDNA fragments from microdissected human chromosome band.
The strategy of isolating the band-specific expression fragments from a probe pool generated by human chromosome microdissection was reported. A chromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1-32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but can't hybridize to 17q11-12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences, and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.
Zhang
M
M
Institute of Genetics, Fudan University, Shanghai, China.
Yu
L
L
Hu
P R
PR
Bi
A D
AD
Xia
J H
JH
Deng
H X
HX
Zhao
S Y
SY
D015152:Blotting, Northern; D001853:Bone Marrow / Q000378:metabolism; D002871:Chromosome Banding; D002883:Chromosomes, Human, Pair 14; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification*; D015723:Gene Library; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D018076:DNA, Complementary