Dna cloning & genetic engineering
DNA cloning requires five main steps: 1) cutting DNA at specific locations, 2) selecting a replicable DNA fragment, 3) joining DNA fragments, 4) inserting recombinant DNA into host cells, and 5) identifying host cells containing the recombinant DNA. Restriction endonucleases and DNA ligases are used to cut and join DNA fragments for cloning. Plasmids and bacteriophages are common cloning vectors used to replicate and amplify recombinant DNA in host cells such as E. coli.
In this document
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Introduction to the Department of Biochemistry and the presenter, Rajesh Chaudhary.
Overview of five key procedures in DNA cloning including cutting, selecting, joining, transferring, and identifying host cells.
Definition of recombinant DNA technology (genetic engineering) used for cloning tasks.
Role of restriction endonucleases and DNA ligases in producing recombinant DNA.
Details on how restriction endonucleases cleave DNA and how DNA ligase joins DNA fragments.
Types and functions of endonucleases: Type I, II, and III, with their cleavage specifics.
Nature of recognition sites for restriction endonucleases, typically 4-6 bp long and palindromic.
Listing sequences recognized by restriction endonuclease type II.
Explanation of gel electrophoresis for separating DNA molecules by size.
Comparison between sticky ends and blunt ends in DNA cloning.
Definition of polylinkers as inserted DNA fragments with multiple recognition sequences.
Role of cloning vectors in amplifying inserted DNA segments.
Details about plasmids: their structure, size, and introduction methods like transformation.
Description of how transformation is performed to introduce plasmids into E. coli.
Brief mention of DNA cloning.
Application of the cloning vector pBR322 to clone foreign DNA into E. coli.
Key features of pBR322: origin of replication and antibiotic resistance for identification.
Overview of bacteriophage as a cloning vector.
Definition and understanding of complementary DNA (cDNA) in genetic analysis.
Overview of hybrid selection methods.
Methods and importance of constructing a human genomic library.
Concept and importance of transgenic animals in biotechnology.
Introduction to PCR as a technique to amplify DNA and its history developed in 1983.
List of the 11 types of PCR including multiplex and quantitative PCR.
Essential components required for PCR, such as DNA template and primers.
Practical aspects and laboratory setup of conducting PCR.
Conclusion slide by presenter Rajesh Chaudhary.
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Dna cloning & genetic engineering
- 1. Department of Biochemistry,Nobel College, Nepal Saturday, June 11, 2016 Rajesh Chaudhary 1
- 2. DNA cloning requiresfollowing five procedure 1. Cutting DNA at precise locations. 2. Selecting a small molecule of DNA capable of self- replicating. 3. Joining two DNA fragments covalently. 4. Moving recombinant DNA from a test tube to a host cell. 5. Selecting or identifying host cells that contain recombinant DNA. Saturday, June 11, 2016 Rajesh Chaudhary 2
- 3. “ ” The method usedto accomplish these and related tasks are collectively referred to as recombinant DNA technology or more informally, genetic engineering. Saturday, June 11, 2016 Rajesh Chaudhary 3 — Lehninger’s Principles of Biochemistry, 6th. Edition
- 4. Restriction Endonucleases andDNA ligases yield recombinant DNA Saturday, June 11, 2016 Rajesh Chaudhary 4
- 5. • Restriction endonucleasecleaves the DNA at specific site. • DNA ligase helps in joining the cut DNA into a vector. Saturday, June 11, 2016 Rajesh Chaudhary 5
- 6. Because it isprotected by methylation of DNA catalyzed by DNA methylase. Saturday, June 11, 2016 Rajesh Chaudhary 6 The restriction endonuclease along with the DNA methylase sometimes referred to as restriction modification system.
- 7. Types and functionof Endonuclease Type 1, Type II, and Type III Both type I and type III are generally large, multisubunit complexes containing both endonuclease as well as methylase activity. Type I restriction endonuclease cleave DNA at random sites that can be more than 1000 bp from recognition sequence. Type II is simpler, requires no ATP, cleaves phosphodiester bond at the recognition site. Type III cleaves DNA 25 bp from the recognition site. Saturday, June 11, 2016 Rajesh Chaudhary 7
- 8. Recognition site Saturday, June11, 2016 Rajesh Chaudhary 8 Thousands of restriction endonuclease has been identified so far and one restriction endonuclease can identify more than 100 of DNA sequence to be cleaved. Recognition sequence are usually 4 to 6 bp long and are palindromic in nature.
- 9. List of sequencesrecognized by restriction endonuclease type II Saturday, June 11, 2016 Rajesh Chaudhary 9
- 10. Saturday, June 11, 2016 RajeshChaudhary 10 DNA molecule can be separated by size using Gel electrophoresis
- 11. Sticky ends VsBlunt ends Saturday, June 11, 2016 Rajesh Chaudhary 11
- 12. Polylinkers Saturday, June 11, 2016 RajeshChaudhary 12 • Inserted DNA fragments with multiple recognition sequences for restriction endonucleases are called Polylinkers.
- 13. Cloning vector allowsamplification of inserted DNA segment Saturday, June 11, 2016 Rajesh Chaudhary 13 • Circular DNA molecule that replicate separately from a host cell. • Sizes: 5000 – 400,000 bp. • Plasmid incorporate several specialized sequence that that enable them to make use of the cell’s resource for their own replication & gene expression. E. coli
- 14. Plasmids Circular DNA molecularthat replicate separately from the host cells. Size: 5,000 – 400,000 Can be introduced into the bacterial cells by the process called “transformation”. Saturday, June 11, 2016 Rajesh Chaudhary 14
- 15. How transformation isdone? The cells (general E.Coli) and plasmid DNA are incubated together at 0 oC in a calcium chloride solution, then subjected to shock by rapid shifting the temperature to 37 0C to 43 0C. Some of the cells uptake the plasmid DNA. Some species of bacteria, such as “Acinetobacter baylyi”, are naturally competent for DNA uptake and do not require the calcium chloride treatment. Saturday, June 11, 2016 Rajesh Chaudhary 15
- 16. DNA cloning Saturday, June11, 2016 Rajesh Chaudhary 16
- 17. Use of pBR322to clone foreign DNA into E.Coli Saturday, June 11, 2016 Rajesh Chaudhary 17
- 18. Use of pBR322to clone foreign DNA into E.Coli Saturday, June 11, 2016 Rajesh Chaudhary 18
- 19. pBR322 important features Anorigin of replication, ori, a sequence where replication is initiated by cellular enzymes. This sequence is required to propogate the plasmid and maintain it at a level of 10-20 copies per cell. Two genes that confer resistance to different antibiotics (tetR, ampR), allowing the identification of cells that contain the intact plasmid or a recombinant version of the plasmid. Saturday, June 11, 2016 Rajesh Chaudhary 19
- 20. pBR322 important features Severalunique recognition sequences (PstI, EcorRI, BamHI, SalI, PvuII) that are targets for different restriction endonucleases, providing sites where the plasmid can later be cut to insert foreign DNA. Small size (4,361 bp), which facilitates entry of the plasmid into cells and the biochemical manipulation of the DNA. Saturday, June 11, 2016 Rajesh Chaudhary 20
- 21. Bacteriophage cloning vector Saturday,June 11, 2016 Rajesh Chaudhary 21
- 22. Complementary DNA (cDNA) Saturday, June11, 2016 Rajesh Chaudhary 22
- 23. Complementary DNA (cDNA) Saturday,June 11, 2016 Rajesh Chaudhary 23
- 24. Hybrid selection Saturday, June11, 2016 Rajesh Chaudhary 24
- 25. Hybrid selection Saturday, June11, 2016 Rajesh Chaudhary 25
- 26. Construction of humangenomic library Saturday, June 11, 2016 26
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- 30. Polymerase Chain Reaction(PCR) A technique used in the molecular biology to make number of copies of DNA using a single DNA. Developed in 1983 by Kary Mullis. Saturday, June 11, 2016 Rajesh Chaudhary 30 Types of PCR Total types of PCR: 11
- 31. Types of PCR 1. Multiplex PCR 2. Asymmetric PCR 3. VNTR PCR 4. Nested PCR 5. Quantitative PCR 6. Hot-start PCR 7. Touchdown PCR 8. Assembly PCR 9. RT-PCR 10. Methylation-specific PCR Saturday, June 11, 2016 Rajesh Chaudhary 31
- 32. Components of PCR DNA as a template Primers Forward Reverse Taq DNA polymerase dNTP (dATP, dGTP, dCTP, dTTP) Mg2+ ions DMSO (optional) H20 Saturday, June 11, 2016 Rajesh Chaudhary 32
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- 34. PCR in laboratorysetup Saturday, June 11, 2016 Rajesh Chaudhary 34
- 35. PCR in laboratorysetup Saturday, June 11, 2016 Rajesh Chaudhary 35
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