Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.
We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.
Kawasaki
K
K
Medical Electronics Research Department, Hitachi Company Ltd., Tokyo, Japan.
Minoshima
S
S
Kudoh
J
J
Shimizu
N
N
D001483:Base Sequence; D002460:Cell Line; D002892:Chromosomes, Human, Pair 22 / Q000378:metabolism*; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005434:Flow Cytometry; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007146:Immunoglobulin lambda-Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D007127:Immunoglobulin Constant Regions; D007146:Immunoglobulin lambda-Chains; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases