Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes

Colin Collins, Wen Lin Kuo, Richard Segraves, James Fuscoe, Daniel Pinkel, Joe Gray

Research output: Contribution to journalArticle

212 Citations (Scopus)

Abstract

Plasmid libraries enriched in sequences from single chromosome types have been constructed for all human chromosomes. This was accomplished by transferring inserts from the Charon 21A phage libraries constructed by the National Laboratory Gene Library Project into Bluescribe plasmids. Insert material freed by complete digestion of the phage libraries with HindIII or EcoRI was cloned into the corresponding sites in Bluescribe plasmids. The sizes of the Bluescribe library inserts determined by gel electrophoresis range from near 0 to ∼6 kb. Fluorescence in situ hybridization (FISH) with the plasmid libraries showed that all hybridize along both arms of the expected (target) chromosome type with varying intensity. However, the plasmid libraries for chromosomes 1, 4, 9, 11, 16, 18, and 20 hybridize weakly or not at all near the centromeres of the target chromosome types. The libraries for chromosomes 13, 14, 15, 21, and 22 cross-hybridize near the centromeres of all members of this group and hybridize weakly to the short arms of the target chromosomes. FISH with each library allows specific staining of the target chromosome type in metaphase spreads. The signals resulting from FISH with libraries for chromosomes 1, 4, 8, 9, 13, 14, 17, 18, 21, and Y are sufficiently intense to permit analysis in interphase nuclei. Examples of the use of these libraries for translocation detection, marker chromosome characterization, and interphase aneuploidy analysis are presented.

Original languageEnglish (US)
Pages (from-to)997-1006
Number of pages10
JournalGenomics
Volume11
Issue number4
DOIs
StatePublished - 1991
Externally publishedYes

Fingerprint

Human Chromosomes
Libraries
Plasmids
Chromosomes
Fluorescence In Situ Hybridization
Chromosomes, Human, Pair 4
Centromere
Chromosomes, Human, Pair 1
Interphase
Bacteriophages
Pluto
Chromosomes, Human, Pair 14
Chromosomes, Human, Pair 13
Aneuploidy
Metaphase
Gene Library
Genetic Markers
Electrophoresis
Digestion
Gels

ASJC Scopus subject areas

  • Genetics

Cite this

Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes. / Collins, Colin; Kuo, Wen Lin; Segraves, Richard; Fuscoe, James; Pinkel, Daniel; Gray, Joe.

In: Genomics, Vol. 11, No. 4, 1991, p. 997-1006.

Research output: Contribution to journalArticle

Collins, Colin ; Kuo, Wen Lin ; Segraves, Richard ; Fuscoe, James ; Pinkel, Daniel ; Gray, Joe. / Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes. In: Genomics. 1991 ; Vol. 11, No. 4. pp. 997-1006.
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