Cloning vector

Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

Abstract: General methods bacterial strains and cloning vectors enzymes that modify DNA and RNA in vitro amplification of DNA using - the polymerase chain reaction (PCR) and the thermostable Taq DNA polymerase, introduction DNA restriction fragment analysis and preparation, introduction in vitro labeling of probes and filter hybridization plasmid DNA preparation for E. Coli hosts preparation of DNA from Lambda bacteriophage clones subcloning fragments into plasmid vectors and plasmid construction preparation of genomic DNA preparation and analysis of RNA from eukaryotic cells - overview geonomic cloning generatino of cDNA libaries preparation of subtractive cDNA, introduction chain termination sequencing transfection of mammalian cells, introduction basic methods of protein analysis in situ hybridization transgenic mouse preparation detection and in vitro generation of specific mutations in genes and cDNAs, introduction appendices.