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Basics of recombinat DNA technology

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Arunkumar Durairaj

This document discusses recombinant DNA technology and the tools required. It describes the basic steps: 1) selecting and isolating the gene of interest, 2) choosing a suitable vector, 3) inserting the gene into the vector, 4) inserting the recombinant DNA into a host cell, and 5) expressing and multiplying the recombinant DNA. It also lists common tools used, including restriction enzymes, cloning vectors, foreign DNA, host organisms, and linkers. Examples of each tool are provided, along with their functions in recombinant DNA technology.

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D ARUNKUMAR Department of Biotechnology B.Tech IIIrd year
Basics of Rec DNA Technology Steps involved in Rec DNA Technology Tools required for it Advantages
 Gene cloning molecular cloning DNA cloning  Mutliplicaton of same copies of DNA again and again
Prokaryotes system – smaller protein Eukaryotes system – larger protein (To fulfill the smaller protein requirements) yeasts bacteria Sacchromyces cerevisiae Pichia pastoris Hansenula polymorpha High yield , low cost , >50KDa Thermal tolerant ,uptake of unusual carbon source E coli Provide s-s proteins ,easier in PTM
1) Selection and isolation of DNA 2) Selection of suitable vector 3) Introduction of Gene of interest to vector 4) Selection and Insertion in host cells 5) Expression and multiplication of recDNA
Selection : To find a part of DNA which responsible for expression of protein system For Insulin human insulin gene is taken Isolation : To isolate the expression system by enzymatically (Restriction endonuclease ) They are also termed as DNA insert , foreign DNA ,target DNA ,clone DNA It can be done by Gel electrophoresis
They are vehicle of the rec DNA technology which carry the information of DNA over a generation They are self replicating in nature Commonly Plasmids and rarely bacteriophage are used Vector cannot be created
To join the gene of interest and vector DNA by enzymatically Ligase is a most commonly used enzyme in rec DNA
Slight distruption of cell wall and the recombinant molecule was inserted into a suitable host by enzymatically Diferent host system were used
Basics of recombinat DNA technology
Basics of recombinat DNA technology
I. Enzymes II. Cloning vector III. Foreign DNA IV. Host organisms V. Linker and adapter sequences
 Necessary tools in RDNA  Commonly used enzymes are Endonuclease Exonuclease Ligase ENDONUCLEASE To cut DNA at a specific site (recognition site /recognition sequence /restriction site /target site ) In 1962 W.ARBER Found that some of enzyme present in Bacteria which can degrade DNA By inserting phage into a E.coli , found the restricting the growth of PHAGE In 1970 MESELSON &YUAN were isolate true endonuclease
Types i) Type I ii) Type II iii) Type III TYPE I RESTRICTION ENDONUCLEASE cleave one strand of DNA at a specific site Requires Mg2+ ions & ATP for functioning
Most stable cleave both strand at a definite length requires Mg2+ ions & not ATP So, advantageous over a TYPE I Cuts between a palindrome sequence with rotational symmentry
Not used in a gene cloning / rec DNA Technology Its an intermediate between TYPE I & TYPE II
By Two ways Blunt cut i)Cleave both double strand at the same point Staggered cut ii) cleave the double strand at different point protruding ends , formation of sticky ends / cohesive ends ( to pair easily )
Removes a part of a nucleotide Can remove either ends(5’ /3’ ) of a DNA Never produce internal cuts Join two fragments By synthesizing the phosphodiester bonds Called as a molecular glue
Capable to replicate in host organism Called as a cloning vehicle / Earner DNA Developed from Bacillus ,Pseudomonas ,Agrobacterium ,Yeast & Fungi Diff. vector system were used Plasmids , bacteriophage , cosmids , Phasmids For a good cloning vector have single site for cutting They should perform glycosylation Express more quantity of protein in terms of quality & quantity Should have a ori of replication
Lack of post translational modifications Mutations may arise whenever improper addition of segments In wwPDP E.coli is used as vector for 23,462 processing
They carries the recDNA and multiplies within a cell and involves the cell division As a result of cell division large number of recombinated cell are produced  which inturn results in the expression of proteins They are easy to transform Easy to replicate Should interfere against replication of recDNA in host cells
 DNA which have to be cloned is called DNA interest or foreign DNA or target DNA  They are responsible for the expression of protein in recDNA technology  They are made up of certain sequence of nucleotides which are responsible  They may be viral /plant /animal /bacterial DNA part

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Basics of recombinat DNA technology

  • 1. D ARUNKUMAR Department of Biotechnology B.Tech IIIrd year
  • 2. Basics of Rec DNA Technology Steps involved in Rec DNA Technology Tools required for it Advantages
  • 3.  Gene cloning molecular cloning DNA cloning  Mutliplicaton of same copies of DNA again and again
  • 4. Prokaryotes system – smaller protein Eukaryotes system – larger protein (To fulfill the smaller protein requirements) yeasts bacteria Sacchromyces cerevisiae Pichia pastoris Hansenula polymorpha High yield , low cost , >50KDa Thermal tolerant ,uptake of unusual carbon source E coli Provide s-s proteins ,easier in PTM
  • 5. 1) Selection and isolation of DNA 2) Selection of suitable vector 3) Introduction of Gene of interest to vector 4) Selection and Insertion in host cells 5) Expression and multiplication of recDNA
  • 6. Selection : To find a part of DNA which responsible for expression of protein system For Insulin human insulin gene is taken Isolation : To isolate the expression system by enzymatically (Restriction endonuclease ) They are also termed as DNA insert , foreign DNA ,target DNA ,clone DNA It can be done by Gel electrophoresis
  • 7. They are vehicle of the rec DNA technology which carry the information of DNA over a generation They are self replicating in nature Commonly Plasmids and rarely bacteriophage are used Vector cannot be created
  • 8. To join the gene of interest and vector DNA by enzymatically Ligase is a most commonly used enzyme in rec DNA
  • 9. Slight distruption of cell wall and the recombinant molecule was inserted into a suitable host by enzymatically Diferent host system were used
  • 12. I. Enzymes II. Cloning vector III. Foreign DNA IV. Host organisms V. Linker and adapter sequences
  • 13.  Necessary tools in RDNA  Commonly used enzymes are Endonuclease Exonuclease Ligase ENDONUCLEASE To cut DNA at a specific site (recognition site /recognition sequence /restriction site /target site ) In 1962 W.ARBER Found that some of enzyme present in Bacteria which can degrade DNA By inserting phage into a E.coli , found the restricting the growth of PHAGE In 1970 MESELSON &YUAN were isolate true endonuclease
  • 14. Types i) Type I ii) Type II iii) Type III TYPE I RESTRICTION ENDONUCLEASE cleave one strand of DNA at a specific site Requires Mg2+ ions & ATP for functioning
  • 15. Most stable cleave both strand at a definite length requires Mg2+ ions & not ATP So, advantageous over a TYPE I Cuts between a palindrome sequence with rotational symmentry
  • 16. Not used in a gene cloning / rec DNA Technology Its an intermediate between TYPE I & TYPE II
  • 17. By Two ways Blunt cut i)Cleave both double strand at the same point Staggered cut ii) cleave the double strand at different point protruding ends , formation of sticky ends / cohesive ends ( to pair easily )
  • 18. Removes a part of a nucleotide Can remove either ends(5’ /3’ ) of a DNA Never produce internal cuts Join two fragments By synthesizing the phosphodiester bonds Called as a molecular glue
  • 19. Capable to replicate in host organism Called as a cloning vehicle / Earner DNA Developed from Bacillus ,Pseudomonas ,Agrobacterium ,Yeast & Fungi Diff. vector system were used Plasmids , bacteriophage , cosmids , Phasmids For a good cloning vector have single site for cutting They should perform glycosylation Express more quantity of protein in terms of quality & quantity Should have a ori of replication
  • 20. Lack of post translational modifications Mutations may arise whenever improper addition of segments In wwPDP E.coli is used as vector for 23,462 processing
  • 21. They carries the recDNA and multiplies within a cell and involves the cell division As a result of cell division large number of recombinated cell are produced  which inturn results in the expression of proteins They are easy to transform Easy to replicate Should interfere against replication of recDNA in host cells
  • 22.  DNA which have to be cloned is called DNA interest or foreign DNA or target DNA  They are responsible for the expression of protein in recDNA technology  They are made up of certain sequence of nucleotides which are responsible  They may be viral /plant /animal /bacterial DNA part