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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

Topic	Lecture	Resource	Page
Introduction	1
What is genetic engineering?	2
Genes and Genome	3
The flow of genetic information	4
Molecular cloning	5
Basic techniques	6
Basic Techniques of Gene Manipulation-an overview	7
Isolation of genomic DNA	8
Agarose gel electrophoresis	9
Nucleic acid blotting	10
Autoradiography	11
Southern blotting	12
Northern blotting	13
Western blotting	14
Transformation of E. coli , Electroporation	15
Transformation with other organisms	16
Polymerase Chain Reaction (PCR)	17
Cutting and Joining DNA molecules	18
Host-controlled restriction modification	19
Types of restriction and modification (R-M) system	20
Nomenclature of restriction endonucleases	21
Target sites of restriction endonucleases	22
Number and size of restriction fragments	23
Summary of restriction endonucleases	24
DNA modifying enzymes	25
Methods of joining DNA fragments	26
DNA ligase to create covalent recombinant DNA	27
Alkaline Phosphatase	28
Blunt end ligation via linker molecules	29
Adaptors	30
Homopolymer tailing	31
Cloning of cDNA by Homopolymer tailing	32
Plasmids	33
Interconversion of plasmid DNA	34
Effect of ethidium bromide on supercoiling of DNA	35
Phenotypic traits exhibited by plasmids	36
Properties of Conjugative and non-conjugative plasmids	37
Host range of plasmids	38
Partitioning and segregative stability of plasmids	39
Incompatibility of plasmids	40
Purification of plasmid DNA	41
Desirable properties of plasmid cloning vehicles	42
Natural plasmids as cloning vehicles	43
Use of pSC101 for cloning	44
pBR322, a purpose-built cloning vehicle	45
Cloning with pBR322	46
Improved vectors derived from pBR322	47
Runaway plasmid vectors	48
Bacteriophage lambda as cloning vector	49
Replication of phage-lambda DNA in lytic and lysogenic cycles	50
Assignment No.1
Modified lambda phages	51
Steps in cloning with lambda	52
Packaging phage-lambda DNA in vitro	53
Vectors for DNA sequencing: bacteriophage M13	54
Cosmid vectors	55
Modified schemes for cloning in Cosmid vectors	56
Phasmid vectors	57
Bacterial artificial chromosomes (BACs)	58
Yeast artificial chromosomes (YACs)	59
Shuttle and Expression vectors	60
Comparison of different cloning vectors-summary	61
Overview of cloning strategies	62
Genomic DNA libraries	63
LambdaEMBL vectors for genomic library construction	64
Genomic libraries in high-capacity vectors	65
PCR as an alternative to genomic DNA cloning	66
Properties of cDNA	67
cDNA libraries	68
Preparation of cDNA for cloning	69
Improved methods for cDNA cloning	70
PCR as an alternative for cDNA cloning	71
Screening strategies	72
Screening by hybridization	73
Benton and Davis’ plaque lift procedure	74
Probe design	75
Assignment No.2
Chromosome walking	76
Chromosome jumping	77
Screening by PCR	78
Expression cloning	79
Immunochemical screening	80
Immunochemical screening of lambdagt11	81
South-western and north-western blotting	82
Screening by functional complementation	83
Requirement for expression in E. coli	84
Secretion of proteins	85
Protein trafficking	86
Stability of foreign proteins in E. coli	87
Constructing the optimal promoter	88
Optimizing translation initiation	89
Stability of mRNA and codon choice	90
The effect of plasmid copy number	91
Plasmid stability	92
Structural instability	93
Host cell physiology can affect the level of expression	94
DNA sequencing: Benefits and Applications	95
Maxam-Gilbert method	96
Chain termination or dideoxy procedure	97
Modifications of chain terminator sequencing	98
Automated DNA sequencing	99
Sequencing accuracy	100
MID TERM EXAM
DNA sequence databases	101
Mutagenesis	102
Cassette mutagenesis	103
Primer extension: the single primer method	104
PCR methods for site-directed mutagenesis	105
Basic PCR reaction	106
PCR principles and procedure	107
DNA polymerases	108
Primers	109
Degenerate primers	110
Types of PCR	111
Competitor RT-PCR	112
Real-time quantitative PCR (qPCR)	113
Nested PCR	114
Inverse PCR	115
Multiplex PCR	116
RAPD	117
RFLP	118
AFLP-PCR	119
Applications of PCR	120
PCR-Medicine	121
PCR-Forensic sciences	122
PCR-Agricultural sciences and environment	123
PCR-Molecular paleontology	124
Genome mapping	125
Markers for genome mapping	126
Genetic linkage mapping	127
Physical mapping	128
Physical versus linkage maps	129
The use of RFLPs in physical maps	130
STS in physical maps	131
SNPs as physical markers	132
Polymorphic DNA detection in the absence of sequence information	133
AFLPs detection in the absence of sequence information	134
Fluorescence in situ hybridization (FISH)	135
Radiation hybrid (RH) mapping	136
Happy mapping	137
Map integration	138
Sequencing Genome	139
High-throughput sequencing	140
Shotgun sequencing	141
Clone-by-clone sequencing	142
Orthologs and paralogs	143
Comparative genomics of bacteria	144
Comparative genomics of organelles	145
Comparative genomics of eukaryotes	146
DNA Microarray	147
Spotted DNA Arrays	148
Oligonucleotide Chips	149
Applications of Microarray-Microbial gene expression analysis	150
Applications of Microarray-Profiling in human disease	151
Phage Display	152
Screening phage display libraries	153
Applications of phage display	154
Knock Outs & Knock Inns	155
siRNA Technology	156
Applications of siRNA	157
Proteomics	158
Two-dimensional (2-D) electrophoresis	159
Mass Spectrometry	160
Protein Microarrays	161
Cloning in gram –ve other than E. coli	162
Cloning in gram +ve bacteria B. subtilis	163
Cloning in Streptomyces	164
Cloning in Archaea	165
Cloning in S. cerevisiae	166
Transformation of fungi with exogenous DNA	167
Vectors for use in S. cerevisiae	168
Properties of different yeast vectors	169
Promoter system for yeast	170
Multipurpose vectors for use in yeast	171
Cloning of large DNA fragments	172
Deficiencies and advantages of YACs	173
Plant transformation	174
Callus cultrue	175
Cell suspension culture	176
Protoplast culture	177
Regenration of fertile plants	178
Major strategies for transformation	179
Assignment No.3
Topic_180_ Agrobacterium mediated transformation	180
Topic_181_T-DNA tansfer	181
Topic_182_Function of T-DNA gene transfer	182
Topic_183_Disarmed Ti vectors	183
Topic_184_Two plasmid strategy	184
Topic_185_Leaf disc transformation	185
Topic_186_Agrobacterium and monocots	186
Topic_187_Rhizogenes and Ri plasmids	187
Topic_188_Protoplast transformation	188
Topic_189_Particle bombardment	189
Topic_190_Chloroplast transformation	190
Topic_191_In planta transformation	191
Topic_192_Plant viruses as vectors	192
Topic_193_Genetic modification in animals	193
Topic_194_Strategies to transform animal cells	194
Topic_195_Chemical transfection techniques	195
Topic_196_Transfection with polyplexes	196
Topic_197_Transfection with liposomes and lipoplexes	197
Topic_198_Physical transfection techniques	198
Topic_199_Microinjections	199
Topic_200_Selectable markers for animal cell gene transfer	200
Topic_201_Bacterial vectors for animal transfection	201
Topic_202_Vrial vectors for transfection	202
Topic_203_Transgenic mice production	203
Topic_204_Pronuclear microinjection	204
Topic_205_Recombinant retroviruses	205
Topic_206_Transfection ES cells	206
Topic_207_Application of transgenic mice	207
Topic_208_Gene transfer to Xenopus	208
Topic_209_Gene transfer to Fish	209
Topic_210_Ways to exploit recombinant DNA	210
Topic_211_Production of recombinant proteins	211
Topic_212_Biopharmaceuticals approved	212
Topic_213_Transgenic animals and plants	213
Topic_214_Recombinant proteins in plants	214
Topic_215_Metabolic engineering in bacteria	215
Topic_216_Metabolic engineering in plants	216
Topic_217_Herbicide resistance in plants	217
Topic_218_Recombinant virus production	218
Topic_219_Fungal resistance	219
Topic_220_Resistance to bacteria	220
Topic_221_BT an insect resistance gene source	221
Topic_222_Drought resistance transgenic crops	222
Topic_223_Transgenic crops for poor quality soil	223
Topic_224_Biotechnology role in food	224
Topic_225_Transgenic model animals	225
Topic_226_Gene medicine	226
Topic_227_DNA vaccines	227
Topic_228_Gene augmentation therapy	228
Topic_229_Gene therapy in cancer	229
Topic_230_Ethics in gene manipulation	230
Final Examination

Virtual University of Pakistan

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BIO732 : Gene Manipulation and Genetic Engineering

Course Overview

Course Synopsis

Course Learning Outcomes

Course Calendar