Isolation of region-specific cosmids by hybridization with microdissection clones from human chromosome 10q11.1-q21.1.
A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.
Karakawa
K
K
Department of Medical Genetics, Osaka University Medical School, Suita, Japan.
Takami
K
K
Nakamura
T
T
Jones
C
C
Fujita
S
S
Ohta
T
T
Jinno
Y
Y
Niikawa
N
N
Inazawa
J
J
Ariyama
T
T
D001483:Base Sequence; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015246:Deoxyribonuclease EcoRI; D015723:Gene Library; D008040:Genetic Linkage; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D009377:Multiple Endocrine Neoplasia / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D004247:DNA; D015246:Deoxyribonuclease EcoRI