图书简介

Preface; Abbreviations; 1 Basic concept of gene cloning; Definition of genetic engineering; Outline of the process; 2 Enzymes used in genetic engineering; Restriction endonuclease; DNA polymerase:; DNA Pol I, Klenow fragment; T4 DNA polymerase (Filling in and Trimming back); Thermostable DNA polymerases; Terminal deoxynucleotidyl transferase; RNA Polymerase; Reverse transcriptase; Alkaline phosphatase; Polynucleotide kinase; DNA ligase:; E. coli DNA ligase; T4 DNA ligase; Deoxyribonuclease; Ribonuclease; 3 Restriction endonucleases; Definition; Discovery; Classification: Type I, II and III; Nomenclature; Recognition sites:; Palindrome, Two-fold axis of symmetry; Specificity and mode of action ; Cognate methylases; Restriction enzymes in genetic engineering:; Sticky end cutters (definition, examples and advantages); Blunt end cutters (definition, examples and advantages); Rare base cutters (definition, examples and advantages); Isoschizomers (definition, examples and advantages); Heterohypekomers (definition, examples and advantages); List of restriction enzymes (nomenclature; recognition; cleavage sites); Unit of restriction enzyme activity; Restriction digestion: partial and complete digestion; Star activity; Restriction digestion vs mechanical shearing method of generation of DNA fragments; Application of restriction enzymes in restriction mapping; 4 Chemical synthesis of oligonucleotides; Why and when opt for chemical synthesis?; Methods of chemical synthesis (details of the process; efficiency; advantages/ disadvantages):; Phosphodiester method; Phosphotriester method; Phosphoramidite method; Photolithographic method; 5 cDNA synthesis; Isolation of RNA and purification of mRNA; cDNA vs genomic DNA; Role of various activities of reverse transcriptase; Various methods for first and second strand DNA synthesis (use of oligo-dT primers, random primers in first strand synthesis; use of replacement method, asymmetrically-tailed plasmid, oligonucleotide primers, random primers in second strand synthesis); RT-PCR; 6 Subfractionation of DNA fragments; Conventional agarose gel electrophoresis (principle; use of LMP; elution from gel); Polyacrylamide gel electrophoresis (principle); Pulsed Field Gel Electrophoresis (PFGE) (principle; variants); Density gradient centrifugation; Gel filtration chromatography; 7 Polymerase Chain Reaction; Why and when use PCR?; Principle of PCR; Variants of PCR:; DD-RTPCR; Degenerate PCR; Asymmetric PCR; Inverse PCR; Anchored PCR; Real time PCR; Scorpion PCR ; RT-PCR; Error prone PCR ; Applications of PCR [Isolation of gene; PCR product cloning (TA cloning, Topo cloning); PCR based mutagenesis; cDNA synthesis; Rapid Amplification of cDNA Ends (RACE); PCR based molecular markers]; 8 Plasmids; Biology of plasmids:; Definition; Why is plasmid not considered genome?; Plasmid size range; Plasmid shapes: covalently closed circular, linear plasmids; Plasmid classification on basis of phenotypic traits: Cryptic plasmid, Fertility (F)/ Conjugative plasmid ; Resistance (R) plasmid (mode of action of resistance genes) Bacteriocinogenic plasmid (mode of action of toxins), Degradative plasmid (mode of action of degrading genes), Virulence plasmid; Mobilizable plasmid; Plasmid host range; Relaxed and stringent control of plasmid copy number; Genome map of Col E1 plasmid; Plasmid as cloning vector (recombinant plasmids):; Advantages of using plasmid vectors; Examples of plasmids: pBR322 (vector map; strategy of construction), pUC (vector map; strategy of construction); 9 Lambda phages; Biology of y phage:; Genome map of y phage; Functions of various gene products; Lysogeny and lytic cycle; Importance of cos sites; y phage as cloning vector:; Advantages of using phage lambda vector; Insertional and replacement vectors (definition; advantage/ disadvantage; examples; vector maps of important vectors); Importance of some terms associated with cloning in y vector:; Insert capacity and packaging constraint, Temperature sensitive repressor, hfl A mutant host Amber mutations in lytic genes, rec A- host, Spi phenotype; 10 M13 phages; Biology of M13 phage:; Genome map of M13 phage; Functions of various gene products; Phage assembly; Structural and functional differences from y phage; M13 phage as cloning vector:; M13 series of vectors (important examples, geneology, advantages, sister vectors, hosts used); 11 Other vectors; Chimeric vectors:; Cosmid (definition; advantages; single cos and dual cos cosmids; examples); Phagemid (definition, advantages, examples, hosts and helper phages used); Phasmid (definition, advantages, examples); Fosmid (definition, advantages, examples); Tumor inducing plasmid (pTi) from Agrobacterium:; Introduction to Agrobacterium; Application in plant transformation; Genome map of pTi; Functions of various gene products; pTi based vectors: Binary and cointegrate vector system (definition; examples; strategy of construction; advantages / disadvantages); Viral vectors:; Advantages of using viral system as cloning vector; Plant viral vectors; Animal viral vector; Artificial chromosomes:; Bacterial artificial chromosome (BAC); P1-derived artificial chromosome (PAC); Bacillus subtilis, Streptomyces, Pseudomonas based vectors (advantages of cloning in non - E. coli systems); Shuttle vector; Expression vector (examples with vector maps; importance of tissue specific, wound inducible, strong, regulatable, T7 and T3 promoters, Details of CaMV 35S promoter); Fusion vector (Translational and transcriptional fusion vectors; advantages of fusion proteins); Specialist vector (for protein secretion, protein solubilization, purification tags, surface display, production of RNA probes); Advanced gene tagging/ trapping vectors (Gene trap vector, Plasmid rescue vector, Enhancer trap vector, Activation tagging); 12 Yeast cloning vectors; Why is a yeast system required?; Types of yeast vectors:; 2u plasmid; Yeast integrative plasmid (YIp); Yeast episomal plasmid (YEp); Yeast replicative plasmid (YRp); Yeast centromeric plasmid (YCp); Yeast linear replicative plasmid (Ylp); Yeast artificial chromosome (YAC); Other fungal systems; 13 Joining of DNA fragments; DNA ligase; Homopolymer tailing (definition; advantages/ disadvantages); Linkers (definition; advantages/ disadvantages); Adaptors (definition; advantages); 14 DNA delivery methods; Methods for DNA delivery to bacterial system:; Conjugation; Mobilization and Triparental mating; Chemical induction; Preparation of competent cells and transformation by heat-shock or freeze-thaw method; In vitro packaging and natural infection; Transduction; Transfection; Ultrasonication; Virus/ phage mediated genetic transformation; DNA delivery to prokaryotic non - E. coli systems; DNA delivery to yeast and other fungal systems; Microprojectile bombardment; Electroporation; Microinjection; DNA delivery into plants/ animals; Agrobacterium mediated genetic transformation and integration of exogenous DNA into plant genome; Virus mediated genetic transformation; Microprojectile bombardment; Electroporation; Microinjection; Liposome packaging; Protoplast fusion; Other uncommon methods: use of silicon carbide whiskers, ultrasonication; DNA integration into bacterial genome; DNA delivery to prokaryotic non - E. coli systems; DNA delivery to yeast and other fungal systems; 15 Cloning strategies using different vector systems; Library:; Genomic library (construction; calculation for probability to clone the desired DNA fragment); cDNA library (construction; effect of mRNA abundance); Genomic library vs cDNA library (advantages/ disadvantages); PCR product cloning; TA cloning; Topo cloning; RT-PCR (an alternative to cDNA cloning); Strategy for cloning in plasmid vector; Strategy for cloning in y insertion vector; Strategy for cloning in y replacement vector; Strategy for cloning in single cos cosmid vector; Strategy for cloning in dual cos cosmid vector; Strategy for cloning in yeast artificial chromosome; Use of linkers, adapters, homopolymer tailing in cloning; 16 Selection/ Screening of recombinants/ transformants; Preparation of probe DNA (radioactive and non-radioactive labeling by methods as nick translation; end filling; random primer methods); Overview of techniques for recombinant selection and screening; Functional (genetic) complementation (blue-white screening; red-white screening; importance of lac Z’ and sup 4); Nutritional complementation (auxotrophic mutants); Gain of function; Colony hybridization; Plaque hybridization; Southern blotting and hybridization; DNA and RNA dot blot; Zoo blot; Plus-Minus screening; Northern blotting, Reverse Northern blotting; Immunological screening; Western blotting; South-Western blotting; North-Western blotting; Hybrid arrest translation (HART) and Hybrid release translation; (HAT); DNA chip; 17 Other techniques for gene manipulation; Mutagenesis and recombination; Site directed mutagenesis; Transposon mutagenesis; Chemical mutagenesis; Insertional mutagenesis; PCR based mutagenesis; Site-specific recombination; Post Transcriptional Gene Silencing; Antisense RNA technology; RNA interference; Cosuppression; 18 Techniques used in Genomics and Proteomics; Genomics:; Rapid DNA and RNA sequencing techniques: Sanger method, Maxam and Gilbert procedure; Automated DNA sequencing; Pyrosequencing; High throughput Sequencing: Shot gun cloning, Chromosome walking, Clone contig assembly, Annotation; Microarray; Proteomics:; Protein sequencing: N and C terminal sequencing, Edman degradation, Dansyl chloride; 2-D electrophoresis; Multi-D liquid chromatography; Mass spectrometry; MALDI-TOF; Yeast two-hybrid system; DNase I foot printing; Protein Microarray; 19 Applications of genetic engineering; Cloning in plant cells:; Developing insect resistance, disease-resistance, herbicide resistant, salt and submergence stress tolerant plants; Quality improvement; Edible vaccines; Applications in biodiversity conservation; Cloning in mammalian cells:; Therapeutic cloning; Reproductive cloning; Origins of organismal cloning in developmental biology-search on frogs; Nuclear transfer procedures; Cloning of sheep (Dolly) and other mammals; Gene knockout technology; Transgenics; Gene therapy; Stem cell therapy; 20 Public concerns; Safety guidelines of recombinant DNA research (Containment facilities and disposal of radioactive material; Protection from exposure of ultraviolet light, ethidium bromide etc.); Ethical issues and prospects for human cloning; Health and environmental concerns related to genetically modified organisms (GMOs)

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