Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.
We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002875:Chromosomes / Q000502:physiology*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000378:metabolism*; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D007621:Karyotyping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D004274:DNA, Recombinant; D013762:Tetrahydrofolate Dehydrogenase