1. 1 Objectives describe the steps in gene cloning by using plasmid as the vector

2. The steps for cloning: 1) Isolation 2) Splicing 3) Insertion 4) Transformation 5) Screening

3. 3 1. Isolation of DNA (gene) Steps in Gene Cloning 2. Slicing with restriction enzymes 3. Insertion 4. Transformation & amplification (multiplication @ cloning) 5. Screening ; to identify bacterial cell containing recombinant plasmid Probing ; to identify bacterial cell containing recombinant plasmid with target gene

4. 4  isolation of plasmid DNA (from E. coli ) and DNA containing gene of interest - plasmid DNA as cloning vector Isolation ♫ plasmid DNA carries amp R gene and lacZ gene - amp R gene: antibiotic resistance gene - lacZ gene : encode for β-galactosidase 

5. 5 Isolation

6. 6  cut open the plasmid DNA at restriction site which lies within lacZ gene  cut out the DNA into many DNA fragments; some of these fragments carry the gene of interest - by using same restriction enzyme which cut at restriction sites containing same palindromic sequence to produce sticky ends Slicing (cutting / cleave)

7. Insertion

8.  after mixing, the DNA fragments and the cut plasmids form the complementary pairs  they are then joined by DNA ligase  creating a mixture of rDNA molecules

9. ♦ note that the lacZ has become nonfunctional ♦ cannot code for β-galactosidase

10. Transformation  bacteria containing recombinant plasmid CAN’T produce β-galactosidase

11.  the rDNA are reintroduced into the bacteria  bacterial cells are mixed with rDNA in the presence of cold calcium chloride  followed by heating; making the bacterial cell wall permeable to plasmids

12. Transformation

13. 13 Transformation  produce diverse of bacteria : bacteria with recombinant plasmid (containing gene of interest) e.g. gene encode for insulin bacteria with recombinant plasmid (containing gene which encode for other protein) e.g. gene encode for human growth hormone bacteria with NON-recombinant plasmid

14. Screening i.e. detecting the bacteria containing rDNA Procedure 1 Blue-white screening

15. 15 - to identify bacteria containing recombinant plasmid Blue-white screening  bacteria is cultured on medium containing antibiotic (ampicillin) and sugar (X-gal) Observation ONLY bacteria with plasmid grow ; has ampicillin resistance (amp R ) gene

16. 16 X-gal is used to identify colonies bacteria with recombinant plasmids  bacteria colonies WITHOUT recombinant plasmid will stain blue ; β-galactosidase is produced by functional lacZ gene (hydrolyze X-gal to yield blue product)  bacteria colonies WITH recombinant plasmid will stain white ; β-galactosidase is NOT produced because lacZ gene is NON-functional; X-gal is NOT hydrolyzed

17. 17 Probing (Nucleic acid hybridization) - to identify bacteria with recombinant plasmid containing gene of interest (target gene) Probing (Nucleic acid hybridization)  based on base pairing between gene of interest (e.g. gene encode for insulin) and other DNA molecule known as DNA probe (short & single-stranded; labeled with radioactive isotope or fluorescent tag)

18. 18 Probing (Nucleic acid hybridization)  a master plate is prepared STEP 1 - contain colonies of bacteria with recombinant plasmid  in the mean time, DNA probe is prepared STEP 2  nitrocellulose filter paper is placed onto the master plate  the filter paper is pressed against the bacterial colonies on the master plate ; bacterial colonies is transferred onto the filter paper

19. 19  the filter paper is treated with NaOH or heat - to denature (separate) the DNA; double helix DNA  single stranded DNA STEP 3 Probing (Nucleic acid hybridization) STEP 4  DNA probe solution is added to the filter paper - DNA probe will hybridize (base-pair with any complementary bases of single stranded DNA)

20. 20  the filter paper is washed to remove the excess, unbound DNA probe STEP 5  then, the filter paper is laid on X-ray film – autoradiography technique  the film (autoradiograph) is developed Probing (Nucleic acid hybridization) STEP 6  the autoradiograph is compared with the master plate  the colonies which contain gene of interest is identified

21. 21

22. 22 REMIND AGAIN

23. 23 Isolation Slicing

24. 24 Insertion

25. 25 TransformationScreening

26. 26 Master plate Probing STEP 1 DNA probe

27. 27 Probing STEP 2 Contain NAOH or Heat To denature DNA,double to single

28. 28 Probing STEP 4

29. 29 Probing STEP 5STEP 6

30. The steps for cloning: 1) Isolation 2) Splicing 3) Insertion 4) Transformation 5) Screening

39. Screening i.e. detecting the bacteria containing rDNA Blue-white screening

40. 40 Probing (Nucleic acid hybridization) - to identify bacteria with recombinant plasmid containing gene of interest (target gene)