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PMID: 2542900 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

Nucleic acids research ·Vol. 17 ·No. 9 ·1989-05-11 ·Pages 3425-33

Anand R, Villasante A, Tyler-Smith C

Abstract

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.

MeSH Terms

Cell Line Chromosomes Chromosomes, Fungal Cloning, Molecular/methods DNA/genetics DNA Transposable Elements Electrophoresis, Agar Gel/instrumentation,methods Gene Library Genetic Vectors Genome, Human Humans Restriction Mapping Saccharomyces cerevisiae/genetics

Chemicals

DNA Transposable Elements DNA

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Anand R

Department of Biochemistry, University of Oxford, UK.

Villasante A

Tyler-Smith C

References (13)

13 references, click to expand

Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis.
Cell. 1984 May;37(1):67-75 PMID: 6373014
Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.
Science. 1987 May 15;236(4803):806-12 PMID: 3033825
Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.
Cell. 1987 Jul 31;50(3):509-17 PMID: 3607877
A model for the separation of large DNA molecules by crossed field gel electrophoresis.
Nucleic Acids Res. 1987 Aug 11;15(15):5925-43 PMID: 3627974
Genome linking with yeast artificial chromosomes.
Nature. 1988 Sep 8;335(6186):184-6 PMID: 3045566
Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast.
Nucleic Acids Res. 1988 Dec 9;16(23):11091-105 PMID: 3060856
Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.
Nucleic Acids Res. 1984 Jan 25;12(2):1049-68 PMID: 6320114
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
J Mol Biol. 1975 Nov 5;98(3):503-17 PMID: 1195397
Restriction endonuclease analysis of ribosomal DNA from Saccharomyces cerevisiae.
Mol Gen Genet. 1976 Nov 17;148(3):233-41 PMID: 796660
Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.
Cell. 1977 Mar;10(3):509-19 PMID: 403010
Inverted repeated sequences in yeast nuclear DNA.
Nucleic Acids Res. 1980 Oct 24;8(20):4651-69 PMID: 7003542
Cruciform structures in palindromic DNA are favored by DNA supercoiling.
J Mol Biol. 1982 Apr 5;156(2):229-43 PMID: 6283098
Transformation of yeast spheroplasts without cell fusion.
Anal Biochem. 1987 Jun;163(2):391-7 PMID: 3310730

Article Info

Journal

Nucleic acids research

Abbr.

Nucleic Acids Res

ISSN

0305-1048

DOI

10.1093/nar/17.9.3425

Published

1989-05-11

Pages

3425-33

Language

English

Region

England

NLM ID

0411011

PMCID

PMC317785

Subset

External Links

PubMed source DOI: 10.1093/nar/17.9.3425 PMC Full Text View journal details

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Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

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