1. Molecular Techniques Department of Stem Cells & Regenerative Medicine ACECR

2. DNA & RNA

3. DNA is a nucleic acid, made of long chains of nucleotides Nucleotide Phosphate group Nitrogenous base Sugar PolynucleotideSugar-phosphate backbone DNA nucleotide Phosphate group Nitrogenous base (A, G, C, or T) Thymine (T) Sugar (deoxyribose) DNA and RNA are polymers of nucleotides

4. Bases Adenine double ringed = Guanine Thymine single ringed = Cytosine purines pyrimidines

6. Hydrogen bonds between bases hold the strands together: A and T, C and G

7. Untwisting and replication of DNA each strand is a template for a new strand helicase 5 end Parental DNA DNA polymerase molecule 5 3 3 5 3 5 Daughter strand synthesized continuously Daughter strand synthesized in pieces

8. RNA is also a nucleic acid –different sugar –U instead of T –Single strand, usually Nitrogenous base (A, G, C, or U) Uracil (U) Sugar (ribose)

9. DNA molecule Gene 1 Gene 2 Gene 3 DNA strand TRANSCRIPTION RNA Polypeptide TRANSLATION Codon Amino acid

10. DNA of gene Promoter DNA RNA polymerase Elongation Termination Terminator DNA Completed RNA RNA polymerase Growing RNA

11. UCAG U C A G G A C U G A C U G A C U G A C U UUUUUU UUCUUC UUAUUA UUGUUG CUUCUU CUCCUC CUACUA CUGCUG AUUAUU AUCAUC AUAAUA AUGAUG GUUGUU GUCGUC GUAGUA GUGGUG phe leu ile met (start) val UCUUCU UCCUCC UCAUCA UCGUCG CCUCCU CCCCCC CCACCA CCGCCG ACUACU ACCACC ACAACA ACGACG GCUGCU GCCGCC GCAGCA GCGGCG ser pro thr ala UAUUAU UACUAC UAAUAA UAGUAG CAUCAU CACCAC CAACAA CAGCAG AAUAAU AACAAC AAGAAG AAAAAA GAUGAU GACGAC GAAGAA GAGGAG tyr stop his gln asn lys asp glu UGUUGU UGCUGC UGAUGA UGGUGG CGUCGU CGCCGC CGACGA CGGCGG AGUAGU AGCAGC AGAAGA AGGAGG GGUGGU GGCGGC GGAGGA GGGGGG cys stop trp arg ser arg gly First Base Third Base Second Base Virtually all organisms share the same genetic code “unity of life”

12. Techniques molecular biology  Polymerase chain reaction (PCR)  Gel electrophoresis  Macromolecule blotting and probing  Southern blotting  Northern blotting  Western blotting

13. Polymerase Chain Reaction

14. Invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993.

15. PCR Reaction Components Template: previously isolated and purified. Two primers: to flank the target sequence)18-28 nucleotides). Four deoxynucleosides triphosphate (dNTPs) : to provide energy and nucleosides for the synthesis of DNA. Buffer system containing magnesium. DNA polymerase.

16. Thermal Cycler

17. PCR Cycles

18. Denaturation

19. Annealing

20. Elongation

21. As amplification proceeds, the DNA sequence between primers doubles after each cycles (The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16…………….)- up to million of times the starting amount until enough is present to be seen by gel electrophoresis. As amplification proceeds, the DNA sequence between primers doubles after each cycles (The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16…………….)- up to million of times the starting amount until enough is present to be seen by gel electrophoresis.

22. Agarose Gel Electrophoresis

23. Since PCR amplifications can generate microgram quantities of product, amplified fragments can be visualized easily following staining with a chemical stain such as ethidium bromide.

24. Variants of PCR Reverse transcriptase-PCR. Nested-PCR. Hot-start PCR. Quantitative PCR. ….

25. Polymerase Chain Reaction: Uses The polymerase chain reaction (PCR) is a technique widely used in: Molecular biology, Microbiology, Genetics, Diagnostics clinical laboratories, Forensic science, Environmental science, Hereditary studies, Paternity testing, and Many other applications…

26. Polymerase Chain Reaction clearly has the potential to become the routine laboratory method for diagnosis of a variety of human disorders : Infectious Diseases: One area where the PCR technique will undoubtedly become a routine method, is the detection of infectious agents, such as pathogenic bacteria, viruses or protozoa. Cancer: Detection of malignant diseases by PCR. Recurrence of hematological cancers has also been evaluated. Detection of micro-metastasis in blood, lymph nodes and bone marrow.

27. Inherited disorders diagnosed using PCR protocols: Alpha1 antitrypsin B thalassaemia Cystic fibrosis Duchenne muscular dystrophy Myotonic dystrophy Haemophilia A and B Huntington`s chorea Phenylketonuria Sickle cell anaemia Familial adenomatous polyposis

28. Comparison of Southern, Northern, and Western analyses

29. ‘Southern’ hybridization named after Sir Edwin Southern Developed in 1975 Southern Blotting

30. Example – Looking for Gene X Cell line 1 2 copies of Gene X extract DNA

31. Example – Looking for Gene X Cell line 2 extract DNA

32. Step 1. Restriction Enzyme Digestion EcoR I

33. Step 1. Restriction Enzyme Digestion

34. Step 2. Gel Electrophoresis _ +

35. _ +

36. TGAATC ACATTG Step 3. DNA Denaturation Eliminate hydrogen bonds with sodium hydroxide (NaOH)

37. Step 4. Transfer DNA to Membrane

38. Step 5. Making a Probe A probe is a small (25-2000 bp) length of DNA or RNA Complementary to the sequence (gene) of interest Labeled for subsequent detection procedures

39. Step 6. Pre-hybridization Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane

40. Step 7. Hybridization

42. Step 8. Washes

43. Step 9. Anti-DIG

45. Step 10. Washes

46. Step 11. CSPD

47. Step 12. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles

48. Step 12. Detection DIG-labeled probes emitting minute amounts of light (chemiluminescence) 32 P-labeled probes emitting ß-particles Autoradiography film can detect this radiation

50. Example – Looking for Gene X Cell line 2 extract DNA 3 Copies

51. Detection of the sickle-cell globin gene by Southern blotting

52. The flow chart of Northern hybridization Prepare RNA samples and run RNA gel Northern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection

54. Any Question ?