Construction of a cDNA library for a specific region of a chromosome using a novel cDNA selection method utilizing latex particles.
A novel method is described for the rapid concentration of particular cDNAs and their mapping to specific regions of a genome. The strategy for 'cDNA scanning' is based on the hybridization of an entire library of cDNAs to a large fragment of genomic DNA that is covalently bound to latex particles. The hybridized cDNAs are eluted, amplified by PCR and cloned into a lambda vector. Selected cDNAs that hybridized to the genomic DNA are cloned, with subsequent sequence analysis. Region-specific DNA fragments prepared from a yeast artificial chromosome (YAC) clone, EG10D9, which maps to chromosome 5 of the small cruciferous plant Arabidopsis thaliana (At), were used to prepare a model system and were covalently bound to latex particles. cDNAs that hybridized to EG10D9 were concentrated by hybridization to the immobilized DNA. The hybridized cDNAs were recovered and amplified by PCR. The resultant sub-library of cDNAs of 0.5-2 kb in length was enriched about 1000-fold. The partial sequences of the cDNAs provided information about genes that are located on the EG10D9 region of the At genome. The cDNA scanning strategy provides an efficient method for the mapping of expressed genes which could be used as expressed sequence tags (EST) within a genome.
Laboratory of Plant Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Tsukuba, Japan.
N
N
Hayashida
Y
Y
Sumi
T
T
Wada
H
H
Handa
K
K
Shinozaki
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D007840:Latex; D008863:Microspheres; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011109:Polymethacrylic Acids; D011137:Polystyrenes; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D007840:Latex; D011109:Polymethacrylic Acids; D011137:Polystyrenes; C042535:polyglycidyl methacrylate