Course No. BBP 155

Course title Principles of genetic engineering and recombinant DNA technology

Number of credits (Lectures-tutorials) 3 (1.5-1.5)

No. of lectures-tutorials 21-21

Course coordinator(s) Dr Anandita Singh

Course outline

The aim of this core-course is to acquaint the students to versatile tools and techniques employed in genetic engineering and recombinant DNA technology. A sound knowledge on the methodological repertoire forming the bases of genomics research is necessary before the students can appreciate how these are innovatively employed in Plant Biotechnology.

Introduction to basic principles of genetic engineering will be provided spanning across topics relating to extra-ordinary power of recombinant DNA enzymes, cloning strategies and bacterial transformation methodologies. Introduction to markers and reporters will be provided in light of their utility for selection and screening of recombinants and/or recombinant transformants. Students will also be introduced to prominent nucleic acid labelling techniques. Introduction to various types of vectors viz. cloning, transformation, expression; and also vectors for genomic and cDNA library and genome sequencing will be provided. A critical appraisal of multifarious sequencing strategies and methods for site-directed mutagenesis will also be covered.

This course may be deemed as a foundation course serving as a platform for introduction of more advanced cutting-edge technologies that essentially are an amalgamation of basic techniques combined in diverse forms and sequence; to be introduced later in the program.

Evaluation procedure

§  2 minor tests 30% each

§  1 major test (end semester) 40%

Details of course contents and allotted time
S.No. / Topic / Allotted time
(hours) /
L / T /
1. / Restriction Endonucleases and other DNA Modifying Enzymes:
Restriction modification system,
DNA modifying enzymes including DNA polymerases, RNA polymerases, Nucleases, Kinases, Phosphatases, Ligases and Reverse transcriptases / 3 / 3
2. / Cloning vectors: Plasmids, Lambda phages, single stranded DNA vectors (M13, fd, f1); Cosmids, Phasmids and Phagemids, YACs, BACs, PACs
Plant Transformation vectors: Introduction to Ti, Ri plasmids and BIBACs
Expression Vectors for high level protein expression / 4 / 4
3. / Cloning strategies: Vector preparation and diverse cloning strategies viz. blunt end cloning, directional cloning, TA-cloning of PCR products, linkers and adaptors based cloning methodologies / 2 / 2
4. / E. coli transformation: Chemical transformation and Electro-poration / 1 / 1
5. / Selection and screening of recombinant transformants: Introduction to marker and reporter genes and selection strategies / 1 / 1
6. / Labelling and detection of nucleic acid sequences:
End-Labelling (3’- and 5’-), Random priming and Nick translation using radioactive non-radioactive labelling techniques / 2 / 1
7. / Genomic DNA libraries: Procedures for Partial, Representative, Enriched, Large-insert DNA libraries, Half-arm cloning
cDNA libraries: Prominent Adapters/Linkers based directional cloning / 2 / 3
8. / Nucleic acid sequencing methodologies and Dye chemistries:
Classical Sequencing methodologies: Maxam and Gilbert’s Chemical cleavage method, Sanger’s Di-deoxy Chain termination method
Dye chemistries for di-deoxy method adapted for automated sequencing
Sequencing methodologies in post-genomic era viz. Pyrosequencing and other cutting edge technologies for high throughput sequencing / 3 / 3
9. / Site Directed Mutagenesis: Cassette mutagenesis, Primer extension (single primer method), PCR methods for site-directed mutagenesis, selection of mutant peptides by phage/plasmid display) / 3 / 3
Total / 21 / 21

The course has been reviewed and commented on by the following experts.

Dr J S Virdi, Reader, Department of Microbiology, University of Delhi, South Campus

Dr Prem Jauhar, Professor of Cytogenetics, USDA-Agricultural Research Service, Northern Crop Science Laboratory, State University Station, North Dakota, USA

Dr Surekha Katiyar Agarwal, Lecturer, Department of Plant Molecular Biology, University of Delhi South Campus

Suggested readings

Principles of Genetic Manipulation Authors: Primrose SB, Twyman RM and Old RW (2001) Blackwell Science Ltd, MA 02148-5020, USA

Molecular Biotechnology: Principles & Applications of Recombinant DNA Authors: Glick BR and Pasternak JJ (2003) ASM Press

Principles and Techniques of Biochemistry and Molecular Biology

Edited by: Keith Wilson and John Walker (2005) CPL Scientific Publishing Services Limited

An Introduction to Genetic Engineering Edited by: Desmond S. T. Nicholl Cambridge University Press February 2002

An Introduction to Molecular Biotechnology: Molecular Fundamentals, Methods and Applications in Modern Biotechnology Edited by: Michael Wink (2006) Wiley