SRM University Department of Bioinformatics Lesson paln Subject : BI0301 Recombinant rDNA technology Class : III Year B Tech Bioinformatics Hrs/week : 4 hrs Name of Staff: Lilly M Saleena
Lecture Hour 1
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Topic Molecular tools for gene cloning Restriction enzymes-host controlled modifications-restriction modification Types-nomenclature- 4, 6 and 8 cuttersfrequency of occurrence of restriction sitesstar activity of restriction enzymes palindromic and non-palindromic recognition sequences - isoschizomersenzymes producing cohesive ends and blunt endsenzymes producing 5’ and 3’ overhangsrecleavable filled-in 5’ overhangs-enzymes producing compatible cohesive ends Dam, Dcm and CpG methylation sensitivity of restriction enzymesDNA and RNA polymerases – thermostability-fidelity-strand displacementnick translation-template specificity (DNA/RNA polymerase)Product specificity-E coli DNA polymerase, T4 DNA polymerase, Klenow fragment (3’ to 5’ exo deleted), Taq DNA polymerase, Pfu DNA polymerase DNA/RNA modifying enzymes-methylasesCpG methylase (M.Sss I), dam methylase
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EcoR I. Ligases – Ecoli DNA ligase, T4 DNA ligase, T4 RNA ligase
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Kinases - T4 polynucleotide kinase. Phosphatases- Topoisomerase
UNIT II 13
Introduction to plasmids- copy
Learning outcome •
Understanding Restriction enzymes and their applications in rDNA technology
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Understanding the principle behind various enymes used in r DNA tchnology
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Understanding plasmids and
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15 16 17 18 19 20 UNIT III 21 22 23 24 25 26 27* 28* UNIT IV 29* 30* 31* 32* 33* 34 35
number,conjugation, compatbility , Plasmids to Vectors, Nomenclature of plasmid cloning vectors , The pedigree of pBR322 and useful properties, pUC18 – a lac selection plasmids Basic features of bacteriophages, structure and organization of Y DNA and M13 phages Cloning vectors based on m13 bacteriophages Cloning vectors based on Y phages – Insertion and replacement vectors Cosmids and phasmids Plasmids in yeast -2um plasmid-yeast based plasmids The structure and use of YAC vectors, The strucrute and use of BAC vectors Cloning techniques What is gene cloning and steps involved Cloning restricted digested product – cohesive end and blunt end ligation Adapters, linkers and homopolymer tailing Creation of restriction sites by PCR TOPO cloning – TA and blunt end cloning Cloning PCR products using SfrI restriction enzyme Transformation techniques Screening techniques Construction of gene liraries PCR – principle and methods PCR types Real time PCR and quatification methods Blotting techniques RFLP and DNA sequencing Construction of cDNA Library by Directional Cloning Construction of Subtractive cDNA Library
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Single Cell cDNA library Speciailized Library for Non-coding RNAs (NcRNAs)
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Construction of Genomic DNA Library
UNIT V
Expression of Recombinant proteins in E
bacteriophages •
Use and types of vectors in rDNA techology
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Learning the principle behind cloning and getting to know about recent advancement in cloning
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Learning the principle behind PCR and getting to know about the different types of PCR
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Learning the principle and procedures about cloning libraries
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coli Plasmid expression vectors-general features promoters used in expression vectors cloning of genes in correct reading frame in expression vectorStrategies for protein secretion purification of recombinant protein using Histidine tag, purification of recombinant protein GST tag, chitin binding domain and intein Codon use in different organisms-codon usage database-codon optimization to increase the expression of recombinant protein.
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Knowing about expression vectors and purification of proteins after expression
TEXT BOOKS 1. Sandy B. Primrose, Richard M. Twyman, Principles of gene manipulation, Blackwell Publishing, 2001. 2. Sambrook and Russell, Molecular Cloning – A Laboratory Manual, CSHL Press, 2002. REFERENCE BOOKS 1. Jerney D. Wale and Malcolm Von Schants, From Genes to Genomes: Concepts and Applications of DNA Technology, John Wiley & Sons, 2002. 2. T.A. Brown, Gene cloning –An Introduction, Nelson Thornes Limited, 1998.
