Microdissection-mediated selection of chromosome region-specific cDNAs.
K562 is a cell line with two acrocentric marker chromosomes containing abnormally banded regions (ABRs), derived from a Ph-positive chronic myelogenous leukemia (CML) patient. Using reverse and forward chromosome painting FISH analysis, we found that 9q34, 13q31, and 22q11 regions co-amplified in the ABRs-bearing acrocentric marker chromosomes of K562. Utilizing the ABRs of the cell line as target DNA for cDNA selection, we established a simple procedure for chromosome region-specific cDNA isolation. After first strand cDNA synthesis from fetal brain mRNAs, short fragment cDNAs (sf-cDNAs) were synthesized with a two-step amplification system by use of our modified Degenerate Oligonucleotide Primed Shuttle Polymerase Chain Reaction (DOP-Shuttle-PCR) method. The sf-cDNAs were hybridized onto RNase A treated metaphases from K562, and the ABRs were microdissected and reamplified with DOP-Shuttle-PCR primer-II. The reamplified sf-cDNAs were cloned into a pBluescript vector. Twenty randomly chosen clones were sequenced and classified into 8 groups. Three out of the 8 grouped clones had been mapped to the long arm of chromosome 22 (22q11), whereas the other 5 were novel cDNAs. Quantitative Southern blot analysis indicated that 7 out of the 8 grouped clones (87.5%) were derived from the co-amplified regions.
Yokoyama
Y
Y
Center for Molecular Biology and Cytogenetics SRL, Inc., Hachioji, Tokyo, Japan.
Ohsugi
K
K
Kozaki
T
T
Sakuragawa
N
N
D001483:Base Sequence; D002874:Chromosome Mapping; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D005819:Genetic Markers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D005819:Genetic Markers; D016044:Fusion Proteins, bcr-abl