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:
2µ 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)
