A region-specific microdissection library for human chromosome 2p23?p25 and the analysis of an interstitial deletion of 2p23.3?p25.1

Abstract

A region-specific library for human chromosome 2p23-p25 was constructed using microdissection and polymerase chain reaction (PCR)-mediated microcloning techniques. This library is large, comprising 300,000 recombinant microclones. The insert sizes range between 50-600 base pairs (bp) with a mean of 200 bp. About 50%-60% of the clones contain unique or very low copy number sequence inserts as determined by their weak or no hybridization to total human DNA. A subset of 48 microclones that did not hybridize to total human DNA after colony hybridization was analyzed, and 26 (54%) clones were shown to contain single-copy inserts and hybridize to human chromosome 2 DNAs, indicating that they are human chromosome 2 specific. The human genomic fragments identified by these clones after cleavage with HindIII have also been characterized. The single-copy microclones were used to analyze an interstitial deletion in the 2p23.3-p25.1 region--46,XY, del(2) (pter-->p25.1::p23.3-->qter)--previously reported in a patient with severe growth and mental retardation and multiple anomalies. Of the 26 microclones analyzed, 14 clones were mapped to the deletion region. The availability of the 2p23-p25 region-specific library and the probes derived from the library should be valuable for fine structure physical mapping analysis and the cloning of disease-related genes localized to the region. These studies also demonstrate the efficiency with which useful probes can be quickly generated for genome studies and for positional cloning.

Gene identification and DNA sequence analysis in the GC-poor 20 megabase region of human chromosome 21

“…Giemsa-trypsin banded metaphase chromosomes from human lymphoblast cell line GM03714 (46, XX, Cell Repository, Camden, NJ) were used in microdissection of the 21cen-q21 region using our standard techniques (16)(17)(18)(19). Twenty-chromosome fragments were microdissected and pooled in a microdrop of proteinase K solution of 5 nl, which was overlayered with paraffin oil to prevent evaporation.…”

“…The pooled chromosome fragments were digested with proteinase K, extracted three times with phenol, cleaved with either MboI or Sau3A, ligated to an MboI linker-adaptor. All these steps were carried out in nanoliter microdrops visualized through a microscope and aided by a de Fonbrune micromanipulator, as described (16)(17)(18)(19). The ligated sequences were transferred to a 200-l microtube and amplified for 15 cycles by the polymerase chain reaction (PCR) using the 20-mer sequence of the MboI linker-adaptor molecule as primer (16).…”

“…Unique sequence microclones were analyzed for insert size, human genomic HindIII hybridizing fragment size, human origin, and chromosome 21 specificity. In these analyses, Southern hybridization was used in which labeled microclones were hybridized to DNAs from (i) human (GM03714), (ii) human͞ mouse cell hybrid WAV17 containing a single human chromosome 21 or human͞Chinese hamster cell hybrid R451 (strain R451-29c-5) containing a single human chromosome 21 (provided by Carol Jones, Eleanor Roosevelt Institute), and (iii) mouse A9 or Chinese hamster CHO-K1 cells, as described (16)(17)(18)(19).…”

“…'' In this study, we used a different approach to isolating useful genomic probes and potential genes from the 21q11-21 region. Employing our previously developed and improved microdissection and MboI linker-adaptor microcloning techniques (16)(17)(18)(19), we constructed four microdissection libraries specifically for this region and isolated large numbers of unique sequence microclones. We then sequenced the microclones and searched in databases to identify homologies with known genes and cDNAs.…”

A region-specific microdissection library for human chromosome 2p23→p21 and the analysis of an interstitial deletion of 2p21

“…hybridized to blots containing DNAs from (a) human (a lymphoblast cell line, GM03714, Cell repository, Camden, NJ), (b) a human/CHO cell hybrid GM10826B con taining a single human chromosome 2 (Cell Repository), and (c) CHO-K1. as previously described (Yu et al, 1992b;1994). Of 94 single-copy clones tested, 60 clones (64%) exhibited posi tive hybridization to both human and chromosome 2 DNA, indicating that these clones were derived from human DNA and are chromosome 2 specific.…”