M13-102: a vector for facilitating construction and improving quality of M13 shotgun libraries.
A modified vector, M13-102, is described which utilizes the previously reported M13-100 direct selection strategy for shotgun cloning [Guilfoyle and Smith, Nucleic Acids Res. 22 (1994) 100-107]. In these vectors, direct selection replaces the need for phosphatase treatment of vector DNA and is achieved by insertional inactivation of M13 gene X. When not inactivated, the engineered overproduction of the M13 gene X product mediates phage replication repression. M13-102 contains two new additions: (1) a sequence enabling triple-helix-mediated affinity capture (TAC) for purification of linearized vector DNA, and (2) universal primer sequences for wider compatibility with commercial instruments that support fluorescence-based sequencing. Using a biotinylated homopyrimidine oligodeoxyribonucleotide as third-strand probe, TAC is performed on streptavidin-coated magnetic beads [Ji et al., Genetics Analysis: Techniques and Applications 11 (1994) 43-47], and serves as a rapid and efficient alternative to gel purification. To reduce tandem insertions, phosphatase treatment of insert DNA was easily invoked without sacrificing cloning efficiency. The combined capabilities of direct selection, TAC purification and phosphatase treatment of inserts should facilitate library construction and improve overall library quality.
Chen
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Department of Chemistry, University of Wisconsin, Madison 53706-1396, USA.
Johnson
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Severin
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Rank
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Smith
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LM
Guilfoyle
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RA
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D004247:DNA; D017931:DNA Primers; D005456:Fluorescent Dyes; D005822:Genetic Vectors; D015698:Genomic Library; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism
D017931:DNA Primers; D005456:Fluorescent Dyes; C051802:triplex DNA; D004247:DNA; D010744:Phosphoric Monoester Hydrolases