BT020302 RECOMBINANT DNA
TECHNOLOGY
Number of Hours / Week: 4
Credits: 4
Course outcome: The
student is exposed to the basic requirements to perform genetic engineering
experiments. The techniques involved in the preparation and introduction of r
DNA to the host. Applications of r DNA technology. Regulations in carrying out
r DNA experiments.
Module
I
Histroy.
Isolation of genetic material. Modification of genetic material for the
preparation of r DNA- Enzymes for in vitro modification of nucleic acids– Kinases
, Phosphatases, Exonucleases, Endonucleases, Restriction Endonucleases, Site
specific recombinases, topoisomerases, Ligases and Terminal Transferases. Types
and properties of restriction enzymes. Modification of Ends - Adapters,
Linkers, Homopolymer Tailing. Genomic and c DNA library construction. 14
hour
Module
II
Cloning
Vectors- Plasmids and their desirable properties, E coli based vectors- pBR,
pSC, pUC, pGEM3Z. M13vectors mp7, Bacteriophages λ EMBL Cosmids, Phasmid ,
Phagemids with special reference to pBluescript, pLITMUS. In vitro packaging,
phage display. Gateway Cloning, TA cloning. Shuttle Vectors -pCAMBIA,Vectors
for Yeast (YEP, YIP, YRP, YCP,YAC) Artificial Chromosomes- BAC, AC. Viral and
virus derived vectors for animal cells- SV40, Adenovirus vectors, Baculovirus,
lentivirus, poxvirus. Plant vectors - geminivirus, Ti plasmid . 15 hour
Module
III
Introduction of r DNA to host cells-micro injection,
electroporation, biolistics, Gene transfer by Chemical transfection: Calcium
phosphate mediated, Polyplexes mediated, Liposomes and lipoplexes mediated.
Markers in prokaryotes. Selection of recombinants. Blue white screening,
screening for Antibiotic resistance. Genetic markers in plants- Kanamycin,
neomycin,
Hygromycin B, Bromoxynil, Methotrexate, chloramphenicol. Animal markers:
Maximizing protein expression in Bacteria, fungi and animal cells – Promoters-
Ca MV promoter, Maize actin 1 gene. Reporter systems- lux genes, GFP.
Expression vectors, Fusion tagged expression system, affinity tag. Studying the
translation product- hybrid arrest and hybrid release translations,
immunochemical methods. Nuclear transfer technology, Inducible expression
system and control of transgene expression through naturally inducible
promoters – lac and tet. Steroid hormones as heterologous Inducers. Chemically
induced dimerisaion (CID) as inducible transgene regulation. Site specific
recombination for efficient gene targeting. 16hrs
Module
IV
Chemical
synthesis of DNA, Blotting techniques: Southern, Northern, Southwestern, Far
western. colony hybridization PCR types and applications. DNA foot printing,
finger printing, gel shift analysis, DNA microarray, RFLP, RAPD, advanced
molecular markers, chromosome walking, jumping and landing. DNA sequencing-
Maxam and gilbert, enzymatic method, pyrosequencing, New generation sequencing-
Site directed Mutagenesis: methods. 16hrs
Module
V
Applications
of recombinant DNA technology- Production and purification of recombinant
proteins- insulin and somatostatin. Gene therapy. Metabolite engineering.
Imparting new agronomic traits to plants to improve quality and quantity. Gene
Silencing through RNA interference and antisense therapy. CRISPR-CAS 9 system.
Gene Knockout. Animal pharming, nanoparticles for labeling, delivery of drugs,
DNA and RNA. Bioethics: laws, possible hazards and merits to society or nature.
15
Reference
1.
Principles of gene manipulation and Genomics – Twyman R M and Primrose S B,
Blackwell pub.ISBN:1405135441
2.
Gene cloning and DNA analysis – T. A. Brown, Blackwell Publishing ISBN:
1405111216
3.
DNA science : A first course in rDNA Technology: David Mickols, Carolina
Biological Supply.
4.
Molecular cloning : A laboratory Manual, Sambrook and Russel, Cold spring
Harbor Lab Pub
5.
Biotechnology: An Introduction, Susan R Barnum. Pub: Thomson, Brooks/Cole
6.
Molecular Biotechnology – B. R. Glick and J J. Pasternac Pub: American Society
for Microbiology
7. From gene to genomes:Concepts and Applications of DNA
Technology – J W Dale, M . Shantz, Wiley- Blackwell
