Isolation of giant DNA fragments from flow-sorted human chromosomes.
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 Alu 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 Alu-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).
Minoshima
S
S
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
Kawasaki
K
K
Fukuyama
R
R
Maekawa
M
M
Kudoh
J
J
Shimizu
N
N
D002461:Cell Line, Transformed; D002875:Chromosomes / Q000032:analysis*; D002891:Chromosomes, Human, Pair 21; D002892:Chromosomes, Human, Pair 22; D003582:Cytogenetics / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D006801:Humans; D008297:Male
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases