Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.
Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Hirota
T
T
Department of Pediatrics, Nagasaki University School of Medicine, Japan.
Tsukamoto
K
K
Deng
H X
HX
Yoshiura
K
K
Ohta
T
T
Tohma
T
T
Kibe
T
T
Harada
N
N
Jinno
Y
Y
Niikawa
N
N
D000015:Abnormalities, Multiple / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D002889:Chromosomes, Human, Pair 2; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D015826:Langer-Giedion Syndrome / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014849:Waardenburg Syndrome / Q000235:genetics