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BIO732 : Gene Manipulation and Genetic Engineering

Course Overview

Course Synopsis

This course will enable students to understand the principle theories of recombinant DNA technology, advanced techniques for working with recombinant DNA, and the applications of the science being taught. This course is generally enlighten and familiarize the students to the hands-on lab practices, working with DNA, RNA by practicing molecular cloning and site- directed mutagenesis, measuring gene expression, and mutation analysis.

Course Learning Outcomes

Recombinant DNA Technology

  • Learning outcomes of this course will be DNA extraction, RNA extraction, quantification of DNA and RNA, PCR nitty-gritties, gel electrophoresis, trouble shooting, recombinant DNA technology and techniques, gene and protein expression methods and techniques, restriction enzymes digestion, blotting techniques, transformation/transfection, electroporation, plasmid, plasmid extraction and expression systems

Course Calendar

What is genetic engineering?2
Genes and Genome3
The flow of genetic information4
Molecular cloning5
Basic techniques6
Basic Techniques of Gene Manipulation-an overview7
Isolation of genomic DNA8
Agarose gel electrophoresis9
Nucleic acid blotting10
Southern blotting12
Northern blotting13
Western blotting14
Transformation of E. coli , Electroporation15
Transformation with other organisms16
Polymerase Chain Reaction (PCR)17
Cutting and Joining DNA molecules18
Host-controlled restriction modification19
Types of restriction and modification (R-M) system20
Nomenclature of restriction endonucleases21
Target sites of restriction endonucleases22
Number and size of restriction fragments23
Summary of restriction endonucleases24
DNA modifying enzymes25
Methods of joining DNA fragments26
DNA ligase to create covalent recombinant DNA27
Alkaline Phosphatase28
Blunt end ligation via linker molecules29
Homopolymer tailing31
Cloning of cDNA by Homopolymer tailing32
Interconversion of plasmid DNA34
Effect of ethidium bromide on supercoiling of DNA35
Phenotypic traits exhibited by plasmids36
Properties of Conjugative and non-conjugative plasmids37
Host range of plasmids38
Partitioning and segregative stability of plasmids39
Incompatibility of plasmids40
Purification of plasmid DNA41
Desirable properties of plasmid cloning vehicles42
Natural plasmids as cloning vehicles43
Use of pSC101 for cloning44
pBR322, a purpose-built cloning vehicle45
Cloning with pBR32246
Improved vectors derived from pBR32247
Runaway plasmid vectors48
Bacteriophage lambda as cloning vector49
Replication of phage-lambda DNA in lytic and lysogenic cycles50
Assignment No.1
Modified lambda phages51
Steps in cloning with lambda52
Packaging phage-lambda DNA in vitro53
Vectors for DNA sequencing: bacteriophage M1354
Cosmid vectors55
Modified schemes for cloning in Cosmid vectors56
Phasmid vectors57
Bacterial artificial chromosomes (BACs)58
Yeast artificial chromosomes (YACs)59
Shuttle and Expression vectors60
Comparison of different cloning vectors-summary61
Overview of cloning strategies62
Genomic DNA libraries63
LambdaEMBL vectors for genomic library construction64
Genomic libraries in high-capacity vectors65
PCR as an alternative to genomic DNA cloning66
Properties of cDNA67
cDNA libraries68
Preparation of cDNA for cloning69
Improved methods for cDNA cloning70
PCR as an alternative for cDNA cloning71
Screening strategies72
Screening by hybridization73
Benton and Davis’ plaque lift procedure74
Probe design75
Assignment No.2
Chromosome walking76
Chromosome jumping77
Screening by PCR78
Expression cloning79
Immunochemical screening80
Immunochemical screening of lambdagt1181
South-western and north-western blotting82
Screening by functional complementation83
Requirement for expression in E. coli84
Secretion of proteins85
Protein trafficking86
Stability of foreign proteins in E. coli87
Constructing the optimal promoter88
Optimizing translation initiation89
Stability of mRNA and codon choice90
The effect of plasmid copy number91
Plasmid stability92
Structural instability93
Host cell physiology can affect the level of expression94
DNA sequencing: Benefits and Applications95
Maxam-Gilbert method96
Chain termination or dideoxy procedure97
Modifications of chain terminator sequencing98
Automated DNA sequencing99
Sequencing accuracy100
DNA sequence databases101
Cassette mutagenesis103
Primer extension: the single primer method104
PCR methods for site-directed mutagenesis105
Basic PCR reaction106
PCR principles and procedure107
DNA polymerases108
Degenerate primers110
Types of PCR111
Competitor RT-PCR112
Real-time quantitative PCR (qPCR)113
Nested PCR114
Inverse PCR115
Multiplex PCR116
Applications of PCR120
PCR-Forensic sciences122
PCR-Agricultural sciences and environment123
PCR-Molecular paleontology124
Genome mapping125
Markers for genome mapping126
Genetic linkage mapping127
Physical mapping128
Physical versus linkage maps129
The use of RFLPs in physical maps130
STS in physical maps131
SNPs as physical markers132
Polymorphic DNA detection in the absence of sequence information133
AFLPs detection in the absence of sequence information134
Fluorescence in situ hybridization (FISH)135
Radiation hybrid (RH) mapping136
Happy mapping137
Map integration138
Sequencing Genome139
High-throughput sequencing140
Shotgun sequencing141
Clone-by-clone sequencing142
Orthologs and paralogs143
Comparative genomics of bacteria144
Comparative genomics of organelles145
Comparative genomics of eukaryotes146
DNA Microarray147
Spotted DNA Arrays148
Oligonucleotide Chips149
Applications of Microarray-Microbial gene expression analysis150
Applications of Microarray-Profiling in human disease151
Phage Display152
Screening phage display libraries153
Applications of phage display154
Knock Outs & Knock Inns155
siRNA Technology156
Applications of siRNA157
Two-dimensional (2-D) electrophoresis159
Mass Spectrometry160
Protein Microarrays161
Cloning in gram –ve other than E. coli162
Cloning in gram +ve bacteria B. subtilis163
Cloning in Streptomyces164
Cloning in Archaea165
Cloning in S. cerevisiae166
Transformation of fungi with exogenous DNA167
Vectors for use in S. cerevisiae168
Properties of different yeast vectors169
Promoter system for yeast170
Multipurpose vectors for use in yeast171
Cloning of large DNA fragments172
Deficiencies and advantages of YACs173
Plant transformation174
Callus cultrue175
Cell suspension culture176
Protoplast culture177
Regenration of fertile plants178
Major strategies for transformation179
Assignment No.3
Topic_180_ Agrobacterium mediated transformation180
Topic_181_T-DNA tansfer181
Topic_182_Function of T-DNA gene transfer182
Topic_183_Disarmed Ti vectors183
Topic_184_Two plasmid strategy184
Topic_185_Leaf disc transformation185
Topic_186_Agrobacterium and monocots186
Topic_187_Rhizogenes and Ri plasmids187
Topic_188_Protoplast transformation188
Topic_189_Particle bombardment189
Topic_190_Chloroplast transformation190
Topic_191_In planta transformation191
Topic_192_Plant viruses as vectors192
Topic_193_Genetic modification in animals193
Topic_194_Strategies to transform animal cells194
Topic_195_Chemical transfection techniques195
Topic_196_Transfection with polyplexes196
Topic_197_Transfection with liposomes and lipoplexes197
Topic_198_Physical transfection techniques198
Topic_200_Selectable markers for animal cell gene transfer200
Topic_201_Bacterial vectors for animal transfection201
Topic_202_Vrial vectors for transfection202
Topic_203_Transgenic mice production203
Topic_204_Pronuclear microinjection204
Topic_205_Recombinant retroviruses205
Topic_206_Transfection ES cells206
Topic_207_Application of transgenic mice207
Topic_208_Gene transfer to Xenopus208
Topic_209_Gene transfer to Fish209
Topic_210_Ways to exploit recombinant DNA210
Topic_211_Production of recombinant proteins211
Topic_212_Biopharmaceuticals approved212
Topic_213_Transgenic animals and plants213
Topic_214_Recombinant proteins in plants214
Topic_215_Metabolic engineering in bacteria215
Topic_216_Metabolic engineering in plants216
Topic_217_Herbicide resistance in plants217
Topic_218_Recombinant virus production218
Topic_219_Fungal resistance219
Topic_220_Resistance to bacteria220
Topic_221_BT an insect resistance gene source221
Topic_222_Drought resistance transgenic crops222
Topic_223_Transgenic crops for poor quality soil223
Topic_224_Biotechnology role in food224
Topic_225_Transgenic model animals225
Topic_226_Gene medicine226
Topic_227_DNA vaccines227
Topic_228_Gene augmentation therapy228
Topic_229_Gene therapy in cancer229
Topic_230_Ethics in gene manipulation230
Final Examination
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