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PCR cloning protocols: from molecular cloning to genetic engineering


, : PCR cloning protocols: from molecular cloning to genetic engineering. PCR cloning protocols: from molecular cloning to genetic engineering: xiv + 490 pp.

This multiauthor text contains practical protocols in the following sections: (1) Performing and optimizing PCR (PCR: basic principles and routine practice, XL PCR amplification of long targets from genomic DNA, Amplification of DNA sequences up to 5 kb from small amounts of genomic DNA using tub DNA polymerase, One-step optimization using touchdown and stepdown PCR, GC-rich template amplification by inverse PCR: DNA polymerase and solvent effects, Coupled one-step reverse transcription and polymerase chain reaction procedure for cloning large cDNA fragments); (2) Cloning PCR products (Using T4 DNA polymerase to generate clonable PCR products, Rapid (ligase-free) subcloning of PCR products, Cloning PCR products utilizing the T/A overhang and a kit, Cloning unmodified PCR products using engineered XcmI restriction sites in a portable cassette, A T-linker strategy for modification and directional cloning of PCR products, Recovery of DNA amplification products from silver-stained polyacrylamide gels: applications in nucleic acid fingerprinting and genetic mapping); (3) Mutagenesis, recombination, and in vitro selection (Recombination and site-directed mutagenesis using recombination PCR, in vitro recombination and mutagenesis of DNA: SOEing together tailor-made genes, In-frame cloning of synthetic genes using PCR Inserts, Creation of chimaeric junctions, deletions, and insertions by PCR, Mutagenesis and gene fusion by megaprimer PCR, Rapid and efficient one-tube PCR-based mutagenesis method, Thermostable ligase-mediated incorporation of mutagenic oligonucleotides during PCR amplification, Linker scanning mutagenesis by three-step PCR, Sequence inversion by flip-PCR, PCR Site-directed mutagenesis using Pyrococcus sp. GBN-D polymerase coupled to a rapid screening procedure: application to a beta -glucanase gene, Using the SELEX combinatorial chemistry process to find high-affinity nucleic acid ligands to target molecules); (4) Cloning unknown neighbouring DNA (Rapid amplification of cDNA ends, Amplification of gene-regulating regions with single-sided specificity, An end-trimming method and its application to amplify adjacent cDNA and genomic DNA fragments by PCR, Anchoring a defined sequence to the 5' ends of mRNAs: the bolt to clone rare full-length mRNAs, Rapid directional walk within DNA clones by step-out PCR, Inverse PCR: an efficient approach to cloning cDNA ends, Rapid amplification of gene ends (RAGE) from gene libraries by anchored PCR, Isolation of coding sequences from yeast artificial chromosome (YAC): clones by exon amplification); (5) Library construction and screening (cDNA libraries from a low amount of cells, Rapid and non-radioactive screening of recombinant libraries by PCR, Use of PCR for cDNA library screening, Generation and PCR screening of bacteriophage lambda sublibraries enriched for rare clones (the "Sublibrary Method")); (6) Differential and subtractive approach by cDNA analysis and cloning (Normalization of cDNA sequence representation by molecular selection, Subtractive cDNA cloning using magnetic beads and PCR, Generation of a PCR-renewable source of subtractive DNA, The use of PCR for differential screening of cDNA libraries, identification and cloning of differentially expressed genes by DDRT-PCR); and (7) Cloning members of gene families (Cloning gene family members using PCR with degenerate oligonucleotide primers, Amplification using degenerate primers with multiple inosines to isolate genes with minimal sequence similarity, Designing PCR primers to amplify specific members of subgroups of multigene families, Screening gene family-enriched cDNA sublibraries with an unamplified cDNA probe: focusing on moderately to abundantly expressed clones).

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