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Aims and Objective The course introduces students to a study of the basic Principles of recombinant DNA technology. This course aims at introducing to students about modern techniques and student is expected to:
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Learning Outcome
Students will be expected to gain an understanding of the techniques in recombinant DNA technology. Upon completion of this course students will be able to describe the principle of molecular biology techniques being utilized in recombinant DNA technology.
Pre-requisites Course
General Biocemistry
Syllabus
DNA cloning methods; cloning vectors including plasmids, bacteriophages, cosmids, YAC vectors, shuttle and expression vectors; gene splicing, genomic libraries, screening methods for gene libraries; DNA cloning restriction enzymes; DNA ligase; tumor inducing (Ti) plasmids; Southern and Northern blotting; chromosome walking; PCR; site specific mutagenesis; production of proteins. Potentials of recombinant DNA technology; gene therapies; restriction fragment length polymorphisms and disease detection (e.g. cystic fibrosis); human genome project; somatic cell cloning, stem cell research, other future prospects; social and commercial considerations.
Assessment Criteria
- Sessional (20): Quizzes/Tests, Assignments, Presentations/ Attendance, Class Participation
- Midterm (30): Subjective (short questions) and Objective Questions
- Final (50): Subjective (short and long questions) and Objective Questions
Time Table
Every Thursday and Friday: 4:00 pm - 5:30 pm
Books Recommended
- Brown T. A., Gene Cloning and DNA Manipulation- An Introduction (2006) 5th Ed., Blackwell Publishmg Inc., USA
- Prinrose S.B., Twyman R.M., and Old R.W. (2006) Principles of Gene Manipulation, 6th Ed., pp. 50-55.
- Tanley W.M.S., and Rapley, Molecular Biomethods Handbook, Chapter 16, Plasmid-Derived Cloning Vectors, Human Press Inc, Totowa. NJ pp, 165-179
- Gene Expression in Recombinant Microorganisms, A. Smith (1995), Marcel Dekker, New York.
- DNA fingerprinting, M. Krawezak and J. Schmidtke (1994), Bioscientific, Oxford
- Principles of Biochemistry, A. L. Lehninger, D. L. Nelson and M. M. Cox, 3rd Editions (2000), Worth Publishers, New York.
- Molecular Biology of the Cell, B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts and J. D. Watson, Third Edition (1994), Garland Publishers
Weekly Course Breakup
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Week |
Lecture |
Topic |
Source (Book) |
Recommendations/Learning Activities |
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1 |
1-3 |
Isolation of DNA, i.e., Total Cell DNA, Plasmid DNA, Phage DNA Media for bacterial DNA Isolation Defined Media Undefined Media Growing and Harveting bacteria Bacetrial Lysis Lysate Preparation |
Book # 2 |
Handouts/Notes |
|
2 |
4-6 |
Chloroform-Phenol Extractions Alkaline lysis DNA Purification Ion Exchange column Purification Cesium chloride density method Concentrating DNA Sample |
Book # 1 |
Class quiz Class Assignment Handouts/Notes |
|
3 |
7-9 |
Plasmid DNA preparation Purification of DNA on basis of Size Purification of DNA on basis of conformation DNA Isolation from Plant Cells CTAB Method |
Book # 1 |
Handouts/Notes Class quiz Class Assignment |
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4 |
10-12 |
Polymerase chain reaction History, reaction, Steps, factors, primer designing, Application of PCR Conditions for pcr reactions DNA Polymerase types and properties Reverse Transcriptase
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Book # 3, 1, 2 |
Presentations |
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5 |
13-15 |
Variants of PCR Real time PCR, Q-PCR TaqMan Probe Molecular Beacons Inverse PCR LM-PCR Ligase chain reaction |
Book # 2, 3, 7 |
Presentations |
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6 |
16-18
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Introduction of DNA Introduction of DNA into Living Cells Purpose of cloning ligated, unligated and self-ligated vector identification Bacterial transformation Competent cells Preparation CaCl2 Method BaCl2 Method
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Book # 1, 7, |
Class quiz Class Assignment Presentations |
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7 |
19-21 |
Selection of Transformed Cells Identification of recombinants Insertional inactivation Antibiotic resistance marker Recombinant selection with pBR322 pUC8 - Lac Z selection Screening for b-galactosidase Blue-White screening |
Book # 1, 2 |
Presentations |
|
8 |
22-24 |
Reporter gene ß-glucuronidase (GUS) (E.coli) green fluorescent protein (GFP) (jellyfish) luciferase (firefly) Inducible expression Nucleic acid probe hybridization |
Book # 1, 2 |
Midterm Exam |
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9 |
25-27 |
Introduction of phage DNA into bacterial cells Introduction of phage DNA into bacterial cell in vitro packaging. Transfection Recombinant λ molecules Packaging systems Phage infection is visualization |
Book # 1, 3 |
Visual Animation Presentations Presentations |
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10 |
28-30 |
Identification of recombinant phages Insertional inactivation of a lacZ′ gene Insertional inactivation of the λ cI gene Selection using the Spi phenotype Selection on the basis of λ genome size Bacteriophage Genomic library cDNA Library |
Book # 1, 2, |
Class quiz |
|
12 |
34-36 |
Cloning Vectors or E.coli simplest cloning vectors - Plamsid Vectors Nomenclature of plasmid cloning vectors pBR322 Properties of pBR322 pBR327 pUC8—a Lac selection plasmid Cloning vectors |
Book # 1, 2, 3, 7 |
Class Assignment Presentations Handouts/Notes |
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13 |
37-39 |
Cloning vectors based on M13 bacteriophage Construct a phage cloning vector Hybrid plasmid–M13 vectors Insertion and replacement vectors 98 Cloning experiments with 2 insertion or replacement vectors Long DNA fragments clone by a cosmid Lambda and other high-capacity vectors enable genomic libraries Vectors for other bacteria
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Book # 1, 2, 3, 8 |
Visual Animation Class quiz Presentations |
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14 |
40-42 |
Cloning Vectors for Eukaryotes Vectors for yeast and other fungi yeast episomal plasmids Other types of yeast cloning vector Artificial chromosomes in yeast The structure and use of a YAC vector Applications for YAC vectors Vectors for other yeasts and fungi Cloning vectors for higher plants Agrobacterium tumefaciens— Ti plasmid |
Book #1, 2, 3, 6 |
Presentations |
|
15 |
43-45 |
Production of transformed plants Cloning genes in plants by direct gene transfer Plant viruses as cloning vectors Caulimovirus vectors Geminivirus vectors Cloning vectors for animals Cloning vectors for insects P elements as cloning vectors for Drosophila Cloning vectors based on insect viruses Cloning in mammals Viruses as cloning vectors for mammals Gene cloning without a vector |
Book # 1, 2, 4 |
Handouts/Notes |
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16 |
46-48 |
Sequencing Genes and Genomes Methodology for DNA sequencing Chain termination DNA sequencing DNA polymerases for sequencing Pyrosequencing Massively parallel pyrosequencing Whole genome Sequencing The shotgun genome sequencing The Haemophilus influenzae genome sequencing project The clone contig approach Clone contig assembly by chromosome walking Clone contig assembly by sequence tagged site content analysis Genetic maps |
Book # 1, 2, 4 |
Handouts/Notes |
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17 |
49-51 |
Gene Expression and Function RNA transcript of a gene – RNA Seq Transcript mapping by hybridization between gene and RNA Transcript analysis by primer extension Transcript analysis by PCR Studying the regulation of gene expression Identifying protein binding sites on a DNA molecule Gel retardation of DNA–protein complexes Footprinting with DNase I Modification interference assays Identifying control sequences by deletion analysis Reporter genes |
Book # 1, 2, 4 |
Handouts/Notes |
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18 |
52-54 |
Applications of Gene Cloning and DNA Analysis in Biotechnology Production of Protein from Cloned Genes - Special vectors for expression Gene Cloning and DNA Analysis in Medicine Production of recombinant pharmaceuticals Identification of genes responsible for human diseases Gene Therapy Gene Cloning and DNA Analysis in Agriculture Gene subtraction Cloning and DNA Analysis in Forensic Science |
Book # 1, 2, 4 |
Handouts/Notes |
Course Material
- Total Lessons 18
- Department Chemistry
- About Visit Profile
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Teacher
Dr Muhammad Fayyaz ur Rehman
