Methods in Gene Cloning

1. 17.3 Methods in Gene Cloning 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. Steps in Gene Cloning 1. Isolation of DNA (gene) 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. Isolation • isolation of plasmid DNA (from E. coli) and DNA containing gene of interest - plasmid DNA as cloning vector • plasmid DNA carries ampR gene and lacZ gene - ampR gene: antibiotic resistance gene - lacZ gene : encode for β-galactosidase 

5. Isolation

6. Slicing (cutting / cleave) • 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

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

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

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. Probing (Nucleic acid hybridization) Probing (Nucleic acid hybridization) - to identify bacteria with recombinant plasmid containing gene of interest (target gene) • 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. Probing (Nucleic acid hybridization) STEP 1 • a master plate is prepared - 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. Probing (Nucleic acid hybridization) STEP 3 • the filter paper is treated with NaOH or heat - to denature (separate) the DNA; double helix DNA  single stranded DNA 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. Probing (Nucleic acid hybridization) STEP 5 • the filter paper is washed to remove the excess, unbound DNA probe • then, the filter paper is laid on X-ray film – autoradiography technique • the film (autoradiograph) is developed STEP 6 • the autoradiograph is compared with the master plate • the colonies which contain gene of interest is identified

22. REMIND AGAIN

23. Isolation Slicing

24. Insertion

25. Transformation Screening

26. Probing STEP 1 Master plate DNA probe

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

28. Probing STEP 4

29. Probing STEP 5 STEP 6

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

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

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