Microdissection of human chromosomal regions 8q23.3–q24.11 and 2q33-qter: Construction of DNA libraries and isolation of their clones

Abstract

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 10 mer DNA linker and 24 mer primer, filling the recessed 3′ ends, and PCR amplification using the 24 mer DNA as a primer. A total of 3.5 × 10 4 pUC19 recombinants (8q library) from the 8q region and 5.0 × 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. Using chromosome fluorescence in situ hybridization, the 4 identical sequences were mapped to 2q33.3 and the 2 overlapping clones to 2q35. These cloned DNAs will be useful in the molecular analysis of TRPS or Waardenburg syndrome type I.