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8.0 RECOMBINANT DNA TECHNOLOGY BY : NUR HIDAYAH MUHAMAD SALEH

8.0 RECOMBINANT DNA TECHNOLOGY BY : NUR HIDAYAH MUHAMAD SALEH. 8.1 RECOMBINANT DNA TECHNOLOGY 8.2 METHODS IN GENE CLONING 8.3 APPLICATION OF RECOMBINANT DNA TECHNOLOGY 8.4 ISOLATING DNA- Experiments. Alba turns green when exposed to blue light. 8.1 RECOMBINANT DNA TECHNOLOGY.

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8.0 RECOMBINANT DNA TECHNOLOGY BY : NUR HIDAYAH MUHAMAD SALEH

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  1. 8.0 RECOMBINANT DNA TECHNOLOGYBY: NUR HIDAYAH MUHAMAD SALEH 8.1 RECOMBINANT DNA TECHNOLOGY 8.2 METHODS IN GENE CLONING 8.3 APPLICATION OF RECOMBINANT DNA TECHNOLOGY 8.4 ISOLATING DNA- Experiments Alba turns green when exposed to blue light

  2. 8.1 RECOMBINANT DNA TECHNOLOGY At the end of this lesson, students should be able to: • Define recombinant DNA technology • Define and explain the tools used in recombinant DNA technology: target DNA (gene of interest), restriction enzyme, DNA cloning vector, host cell and modifying enzymes (DNA ligase).

  3. RECOMBINANT DNA (rDNA) • Recombinant DNA refers to DNA which has been altered by joining together segments of DNAfrom two different sources, generally a different species. • It usually involves putting a gene from one organism into the genome of a different organism to produce new genetic combination that has commercial value or medical purpose. • Formed through laboratory method/ in vitro. • Organism that have had a foreign gene inserted into them are called transgenic organism.

  4. WHAT IS GENE CLONING? • Cloning is the process of creating an identical copy of an original organism or thing. • A cloning results in organism that has been directly copied from and is therefore genetically identical to another living organism. • Gene cloning is a process of producing genetically identical copies of DNA. • In gene cloning, only particular gene is copied.

  5. RECOMBINANT DNA & CLONING

  6. Target DNA (gene of interest) Restriction enzymes DNA cloning vector – Plasmid Host cell – E. coli Modifying enyzmes –DNA ligase TOOLS IN RECOMBINANT DNA TECHNOLOGY

  7. 1) TARGET DNA • Target DNA containing gene of interest • The gene encode for the desired product or introduce desirable traits into an organism

  8. 2) RESTRICTION ENZYMES • Also known as restriction endonuclease. • Class of enzyme that recognizes specific base sequence of DNA and then cut the DNA at that specific base sequences, called restriction site. • Restriction enzymes are named for the organisms from which they are isolated: Escherichiacoli R13 - Eco RI Serratiamarcescens - Sma I • Different restriction enzyme cut a different restriction site.

  9. Restriction site • SAME sequence of bases on both DNAstrands but arranged in opposite direction. • The sequence is said to be palindromic. • A palindrome is a word, phrase, number or other sequence of units that has the property of reading the same in either direction.

  10. Pattern of cutting (Sticky ends vs Blunt ends) • Blunt ends- produced when restriction enzyme cut DNA in a straight way. E.g. SmaI • Sticky ends- produced when restriction enzyme cut DNA in a staggered way. E.g. EcoR1

  11. 3) DNA CLONING VECTOR Lac Z gene  an agent/small DNA molecule that can carry foreign DNA fragment into a host cell and replicate there. • Commonly used vectors include : • Plasmid e.g: pUC 18 • Bacteriophage e.g : λ2001 • Cosmid e.g : sCOS-1 • Yeast Artificial Chromosome (YAC) e.g : pYAC Multiple cloning site (MCS) Antibiotic resistance gene Ori gene

  12. Characteristic of cloning vector • Able to accept/carry foreign DNA in MCS • Must have multiple cloning site (MCS) for cloning. Means they have two or more restriction sites recognized by various restriction enzymes. • Able to replicate freely in the host cell because contain an origin of replication-Ori gene • Able to express cloned genes. • Have selectable genetic marker//antibiotic resistant gene  ampR(ampicillin resistant gene) • Small in size (2.7kb-1.0Mb)

  13. RECOMBINANT DNA

  14. 4) Host cell • An organism that can serve as living host for replication and expression of recombinant DNA (rDNA). • The organism that can act for this function is bacteria (E.coli), animal, plant cell, yeast. • Must able to accept foreign DNA, that is the cloning vectors carrying the target gene, and allows the vectors to multiply.

  15. CHARACTERISTICS OF HOST CELLBACTERIA • Able to receive recombinant DNA through the transformation process • Able to maintain the structure of recombinant DNA from one generation to the next generation. • Able to amplify/ multiply the gene product from recombinant DNA. • Able to express the gene of interest • Relatively larger than DNA cloning vector • Antibiotic sensitive

  16. 5) Modifying enzyme • Join the gene of interest and DNA cloning vector to form recombinant DNA. • Both must have complimentary sticky ends or blunt ends. • An example of modifying enzyme is DNA ligase. • Catalyzed the formation of phosphodiester bond between sugar and phosphate of adjacent nucleotide.

  17. 8.2 METHODS IN GENE CLONING At the end of this lesson, students should be able to: • Overview using diagram to show the steps in gene cloning by using plasmid as the vector.

  18. Steps in gene cloning  using plasmid as vectors • Isolation of gene • Cleave / cut • Insertion • Transformation and Amplification • Screening (Blue/ white screening) METHODS IN GENE CLONING

  19. i) ISOLATION OF GENE • Plasmid (DNA cloning vector) from E.coli b) Target gene (gene of interest) from the donor cells

  20. II) CLEAVE / CUT • Target gene and plasmid are cut using the same restriction enzymeto produce complimentary sticky or blunt ends. • Complimentary base pair allow both DNA to ligate/ join. Target gene Vector Both cut with same restriction enzyme, EcoRI Sticky ends

  21. III) INSERTION • Target DNA is inserted into the plasmid • The complimentary bases will be attached to each other • DNA fragments are then joined by using DNA ligase • Forming recombinant DNA/ recombinant plasmid/ rDNA

  22. IV) TRANSFORMATION AND AMPLIFICATION • Recombinant DNA is transferred into the host cell/ bacteria • By transformation. • The bacteria are cultured in medium containing nutrient agar. • As bacteria cell multiply, the gene of interest will be replicated within each cell. • Recombinant DNA is amplified in the host cell.

  23. IV) TRANSFORMATION AND AMPLIFICATION SOMEHOW.. • Not all the plasmids will have taken up the intended foreign DNA • Not all the bacteria will have taken in plasmid There are 3 possible types of bacteria: • Bacteria that do not take up any plasmid • Bacteria that take up non recombinant plasmid • Bacteria that take up recombinant plasmid **so screening technique is required here.

  24. Target DNA Bacteria with recombinant plasmid Bacteria without any plasmid Bacteria with non recombinant plasmid

  25. V) SCREENING • Screening can be done by blue white screening • The purpose is to identify bacteria cell containing recombinant DNA • Bacteria obtained from amplification steps are cultured in a medium containing: • Ampicillin ( antibiotic) • X-gal (colorless sugar) • Colony of bacteria containing recombinant DNA are then cultured on a large scale.

  26. V) SCREENING

  27. BLUE/WHITE SCREENING WHY AMPICILLIN? • Antibiotic that normally kills the bacteria • Bacteria that take up plasmid will be able to grow and form colonies on the medium because plasmid have ampicillin resistant gene. WHY X-GAL? • The bacteria that contain plasmids with intact lacZ gene (do not carry target gene) are able to produce β- galactosidasewhich can hydrolyze X-gal, produce a compound which make colony blue. • The bacteria which contain recombinant plasmid will form white colonies because the lacZ gene is disrupt and non functional.

  28. Plasmid before and after insertion of target DNA

  29. Medium containing ampicillin and X-gal Cells are resistant to antibiotic and produce white colony because lacZ gene disrupt Cells are sensitive to antibiotic and do not grow Cells are resistant to antibiotic and produce blue colony because lacZ gene intact

  30. 8.3 APPLICATION OF RECOMBINANT DNA TECHNOLOGY At the end of this lesson, students should be able to: • Briefly explain applications of recombinant DNA technology in mass production of insulin using cDNA.

  31. PRODUCTION OF INSULIN

  32. REVERSE TRANSCRIPTION

  33. REVERSE TRANSCRIPTION mRNA  isolated from the pancreas cell  a test tube. mRNA act as a template/ undergoes reverse transcription. Reverse transcriptase and free DNA bases are used to produce single strand complimentary DNA (cDNA) mRNA  digested by certain enzyme. Single strand cDNA acts as template DNA polymerase and free DNA basessynthesize second strand of cDNA The double helix cDNA is now ready to be inserted into the plasmid.

  34. Extract mRNA for insulin from βcells of Islet of Langerhans in pancreas cDNA is produced from the mRNA template using reverse transcriptase. Amplify cDNA using polymerase chain reaction (PCR) Restriction enzyme cuts cDNA at restriction site and creates complimentary sticky ends Isolate plasmids fromE.coli and cuts with the same restriction enzyme Insert target gene (cDNA) into plasmid and are joined using DNA ligase Transformed into bacteria/ E.coli Screening procedure (Blue white screening of the recombinant plasmid) Amplification and extraction of insulin from E.coli

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