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Bt6603-Genetic-Engin BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 DEPARTMENT OF BIOTECHNOLOGY Faculty Name : Ms. K. Ramya Faculty Code : HTS 1277 Subject Name : Genetic Engineering and Genomics Subject Code : BT6603 Year & Semester : III & VI BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS Faculty Name : Ms. K. Ramya Faculty Code: HTS 1277 Subject Name: Genetic Engineering and Genomics Subject Code: BT6603 Department: Biotechnology Year & Semester: III & VI COURSE OUTCOMES On completion of this course, the students will be able to CO No Course Outcomes Knowledge Level Understand about the cloning of commercially important genes and production of C315.1 recombinant proteins K2 C315.2 Understand about the construction and screening of DNA libraries K2 C315.3 Discuss about the gene and genome sequencing techniques K2 C315.4 Explain about the microarrays, analysis of gene expression and proteomics K2 C315.5 Understandarticulate the applications of genome analysis and genomics K2 Mapping of Course Outcomes with Program Outcomes and Program Specific Outcomes BT6602 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3 PSO4 C315.1 2 - - - - - - - - - - 3 - - - - C315.2 2 - - - - - - - - - - - - - - - C315.3 2 - - - - - - - - - - - - - - - C315.4 2 - - - - 3 3 - - - - 3 - - - - C315.5 1 - - - - 3 3 - - - - 3 - - - - BT6602 PO1 PO2 PO3 PO4 PO5 PO6 PO7 PO8 PO9 PO10 PO11 PO12 PSO1 PSO2 PSO3 PSO4 C315 2 - - - - 3 3 - - - - 3 - - 3 - K1 – Remember; K2 – Understand; K3 – Apply; K4 – Analyze; K5 – Evaluate; K6 - Create Mapping Relevancy 1: Slight (Low) 2: Moderate (Medium) 3 Substantial (High) - : No correlation BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 BT6603 GENETIC ENGINEERING AND GENOMICS UNIT-I PART A 1. State four safety guidelines in creating rDNA. i Care should be taken that novel organism created should not be normal ii. Treatment with alkaline phosphatase to increase the number of recombinants iii. Appropriate insert and vector size should be selected. iv. Proper technique for increasing the transformation efficiency should be selected. 2. What are the basic properties of a plasmid vector? (MAY 2014) Low molecular weight, ability to confer readily selectable phenotypic trait on host cells, single sites for a large number of restriction endonuclease preferably in genes with a readily scorable phentotype. 3. What are DNA modifying enzymes? Give one example.(DEC ‘2010) (May’ 2012,2016) These are the enzymes that are involved in the degradation synthesis and alteration of nucleic acids.DNA ligase - An enzyme which seals single stranded nicks between adjacent nucleotides in a duplex DNA chain. Alkaline phosphatase - Removes the 5’ phosphates and replaces it by hydroxyl group. 4. What are isoschizomers and Neoschizomers? Give any two examples.(DEC’2013, 2015) Isoschizomers are the enzymes obtained from different sources but recognizes the same target.Ex-SmaI ,XmaI. Neoschizomers are restriction enzymes that recognize the same nucleotide sequence as their prototype but cleave at a different site. For example:Prototype MaeII A^CGT produces DNA fragments with a 2-base 5' extension Neoschizomer TaiI ACGT^ produces DNA fragments with a 4-base 3' extension 5. If you add ligase to alkaline phosphates treated vector does the ligation takes place? Justify your answer. No. because the phosphate group at 5 end is replaced by hydroxyl group, so phosphodiester linkage is not formed. 6. Define restriction endonuclease.(MAY 2013,2014), (Dec' 2016) Nuclease that recognizes specific nucleotide sequences in a DNA molecule and cleaves or nicks the DNA particular site. 7. State the difference between 3 types of restriction endonuclease Type I – recognizes and cleaves the DNA upto 1000 bp away from the site. Type II – recognizes and cleaves at specific target site. Type III- recognizes and cleaves at 26-30 bp away from the target size. 8. Give few examples for restriction endonuclease. (MAY 2013), (Dec' 2016). Sma I, Hae III, Hind III, Bam HI. 9. Difference between cohesive sticky ends & blunt end. S.NO STICKY END BLUNT END Cut he bases around the center Cut he bases at the of symmetry center of symmetry Promotes effective ligation Ligation efficiency is 2. less BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 Eg.GAATTC → G GAATTC → GAA AATTC TTC 3. CTTAAG CTTAA CTTAAG CTT G AAG 10. Give the steps involved in creating a recombinant plasmid. Isolation of gene of interest, selection of vector, cutting vector with restriction enzyme, joining with DNA ligase, transformation, screening and expression of the cloned genes. 11. Draw a neat diagram of plasmid vector. 12. Which type of restriction enzyme is used for creating rDNA? (May’ 2012) Justify? Type II restriction enzyme because they produce cleavage at or near host specificity site. 13. What are Biotin and Avidin? What is their role in rDNA technology? (DEC’ 2010). Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. Biotin, historically known as Vitamin H is a water-soluble B-complex vitamin (vitamin B7) discovered by Bateman in 1916. It is composed of a ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring. Biotin is a coenzyme in the metabolism of fatty acids and leucine, and it plays a role in gluconeogenesis. Their role in rDNA technology is for tagging purpose. 14. What is RCGM and mention its role (MAY’ 2010) Review Committee on Genetic Modification (India). Monitors research projects safety aspects 15. Differentiate a promoter and an enhancer (MAY’ 2011), (Dec' 2016) Enhancer DNA sequences bind transcription factors called enhancer-binding proteins which increase the rate of transcription. Enhancer sequences may be kilobases away from the gene they influence. An enhancer complex may interact with promoter complexes by bringing the sites into direct contact. Promoter a regulatory region of DNA located upstream of a gene, providing a control point for regulated gene transcription 16. Give a name of a modifying enzyme that helps in converting blunt end DNA to sticky ends (MAY’ 2011, 2012) Terminal transferase 17. What is meant by expression vector? (May’ 2012), (Dec' 2016) Plasmids or phages carrying promoter regions to cause expression of inserted DNA sequences 18. Define Cosmid.(MAY 2013), (Dec' 2016). A vector designed to allow cloning of large segments of foreign DNA. They are hybrids BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 composed of the COS sites of lambda inserted into a plasmid. Helps in joining the 2 DNA fragments. 19. What are the requirements for an efficient prokaryotic expression vector? (DEC’2013), (Dec' 2016) Constructing the optimal promoter, optimizing translation initiation, maintenance of the stability of mRNA, effect of codon size ,transcription termination, plasmid copy number ,plasmid stability and host cell physiology. 20. Differentiate adaptors from linkers.(DEC’ 2010), (MAY’ 2010, 2014)) Linkers are short stretches of double stranded DNA of length 8-14 bp and have recognition site for 3-8 RE. These linkers are ligated to blunt end DNA by ligase. Adapters are linkers with cohesive ends or a linker digested with RE, before ligation. The most widely used definition is cut linkers also called as adapters. 21. What are phagemids? How are they different from cosmids? (DEC’ 2010) A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Many commonly used plasmids contain an f1 ori and are thus phagemids. Phagemids: F1 origin cloned into a plasmid Cosmids: Cos sites cloned into a plasmid 22. Name any two eukaryotic Transcription factors. Give their functions. (MAY 2013), (Dec' 2016) General transcription factors of the pre-initiation complex are required for the expression of all structural genes transcribed by RNA polymerase II (Ex): TFIID → TBP + TFIIA, TFIIB, TFIIF, TFIIE,TFIIH Specific transcription factors bind to proximal promoter DNA sequences or distal enhancer elements. (Ex): homeodomain proteins, p53, etc 23. Name any two special features of pBR 322. (MAY 2013) pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1, the ampR gene, encoding the ampicillin resistance protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). 24. What are the applications of polylinkers. (Dec 2014) A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology, bioengineering, and BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 molecular genetics, MCSs let a molecular biologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs). 25. Write briefly about genetic makeup of T4 phage.
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