Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome.
We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.
Hadano
S
S
Ikeda Genosphere Project, ERATO, JRDC, Tokai University School of Medicine, Kanagawa, Japan.
Watanabe
M
M
Yokoi
H
H
Kogi
M
M
Kondo
I
I
Tsuchiya
H
H
Kanazawa
I
I
Wakasa
K
K
Ikeda
J E
JE
D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods; D002894:Chromosomes, Human, Pair 4 / Q000648:ultrastructure; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D005347:Fibroblasts; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D007621:Karyotyping; D007834:Lasers; D008846:Micromanipulation / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes