An efficient method for selecting unique-sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in situ hybridization.

J C Fuscoe; C C Collins; D Pinkel; J W Gray

doi:10.1016/0888-7543(89)90092-x

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An efficient method for selecting unique-sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in situ hybridization.

J C Fuscoe·C C Collins·D Pinkel·J W Gray

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Abstract

This paper describes an efficient procedure for selecting large numbers of unique-sequence or very low repeat-sequence probes from recombinant phage libraries. Probes were selected from the Charon 21A library LL21NS02 (made from DNA from human chromosome 21) in a multistep process in which (1) inserts from LL21NS02 were subcloned into Bluescribe plasmids, (2) plasmids were grown at high density in colonies on nitrocellulose, and (3) plasmids were selected as containing unique-sequence inserts if DNA from the colonies failed to hybridize, at low stringency, to radiolabeled total human DNA. In this manner, 1530 colonies were picked to form the library pBS-U21/1530. About 80% of the recombinants constituting pBS-U21/1530 were shown by Southern analysis to carry inserts that are present in only one copy in haploid genomic human DNA. Approximately 70% of the sequences mapped to human chromosome 21. Fluorescence in situ hybridization with DNA from pBS-U21/1530 allowed specific, intense staining of the number 21 chromosomes in metaphase spreads made from human lymphocytes.

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Bacteriophage lambda Blotting, Southern Chromosomes, Human, Pair 21 Cloning, Molecular DNA Probes DNA, Recombinant DNA, Viral Electrophoresis, Agar Gel Fluorescence Gene Amplification Genetic Techniques Genetic Vectors Humans Nucleic Acid Hybridization Plasmids Repetitive Sequences, Nucleic Acid Journal Article Research Support, U.S. Gov't, P.H.S.

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J C Fuscoe, C C Collins, D Pinkel, J W Gray. 1989. An efficient method for selecting unique-sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in situ hybridization.. https://doi.org/10.1016/0888-7543(89)90092-x

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