Isolation of giant DNA fragments from flow‐sorted human chromosomes

Abstract

We have established a method using a conventional cell sorter equipped with a single argon laser to sort intact human chromosomes that can be used as a source for the production of giant DNA fragments. Various improvements were made to both the equipment and sorting method to enhance the sorting resolution and avoid destruction of chromosomal DNA. Using this improved method chromosomes 21 and 22 were sorted from the B-lymphoblastoid line GM00130B, digested with the rare cutting restriction endonuclease NotI, and analyzed by pulsed field gel electrophoresis followed by Southern hybridization using the AZu repetitive sequence as a probe. More than 25 discrete NotI giant DNA fragments ranging from 50 kb to longer than 2.5 Mb were separated and the size distribution pattern was unique for each chromosome, indicating successful sorting of intact chromosomes. The cumulative size of these AZu-positive NotI DNA fragments were 22.7 Mb and 25.5 Mb for chromosomes 21 and 22, respectively. These values are 47% and 49% of the estimated size of chromosomes 21 (48 Mb) and 22 (52 Mb).

Detection of variation in the ribosomal RNA gene clusters by a modified fluorescencein situ hybridization method

“…Human diploid lymphoblastoid cell line GM00130B was maintained as previously described (Minoshima et al, 1990). The karyotype of this line appears normal and stable as indicated (Minoshima et al, 1990).…”

“…Human diploid lymphoblastoid cell line GM00130B was maintained as previously described (Minoshima et al, 1990). The karyotype of this line appears normal and stable as indicated (Minoshima et al, 1990). Metaphase chromosome spreads were prepared by the standard colcemid trealment, hypotonic shock and methanol/acetic acid fixation as described (Grill et al, 1985).…”

Gene mapping of human bilirubin UDP-glucuronosyl transferase on 1q21-q23 by a cell sorter andin situ hybridization

“…Preparation of metaphase chromosome suspension was according to Lebo et al (1985) and Minoshima et al (1990) with several modifications. The spontaneously transformed human B-]ymphoblastoid cell lines GM00130B (46, XY) was obtained from the NIGMS Human Genetic Mutant Cell Repository (Camden, N J).…”

The organization of the human immunoglobulin lambda gene locus.

“…A 1.4-Mb NotI fragment containing the )~ gene locus was isolated from flow-sorted human chromosome 22 (Minoshima et al 1990;Kawasaki et al 1992b). Small DNA fragments (-300 bp) were then amplified from the NotI fragment by linker ligation PCR.…”