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

James C. Fuscoe, Colin C. Collins, Dan Pinkel, Joe Gray

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 LL21NSO2 (made from DNA from human chromosome 21) in a multistep process in which (1) inserts from LL21NSO2 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 a allowed specific, intense staining of the number 21 chromosomes in metaphase spreads made from human lymphocytes.

Original languageEnglish (US)
Pages (from-to)100-109
Number of pages10
JournalGenomics
Volume5
Issue number1
DOIs
StatePublished - 1989
Externally publishedYes

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Chromosomes, Human, Pair 21
Human Chromosomes
Gene Library
In Situ Hybridization
Clone Cells
Staining and Labeling
Plasmids
DNA
Libraries
Pluto
Collodion
Haploidy
Metaphase
Fluorescence In Situ Hybridization
Bacteriophages
Lymphocytes

ASJC Scopus subject areas

  • Genetics

Cite this

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. / Fuscoe, James C.; Collins, Colin C.; Pinkel, Dan; Gray, Joe.

In: Genomics, Vol. 5, No. 1, 1989, p. 100-109.

Research output: Contribution to journalArticle

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