Molecular Diagnosis I: Methods in Molecular Medicine

1. Molecular Diagnosis I: Methods in Molecular Medicine 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A705, Research Building 2012/09

2. Application of genomic knowledge & technology will significantly improve the diagnosis and treatment of the disease • General Personalized Trends of Medicine in 21th Century • Treatment afterward Predictive & Preventive • Molecular epidemiology: Sub-population • Rare diseasesCommon diseases

3. Why Clone Human Genes? • To define inherited genetic mutations • Develop diagnostic reagents (pre-natal testing) • Develop targeted therapies • To isolate functional/normal genes • Prepare normal protein products (Factor VIII) • Therapeutic use of proteins normally produced at low levels • Vaccine development (avoid attenuated organisms) • To isolate genes with somatic mutations • Develop diagnostic reagents • Target therapy to altered gene product • To define and understand cellular pathways and circuits • Signal transduction, immunity, development, cell cycle and apoptosis

4. The basic tools of gene exploration • Restriction-enzyme analysis:“molecular scalpels” • Recombinant DNA technique: • Blotting technique: 1.Southern Blot: DNA hybridization 2. Northern Blot: RNA hybridization 3. Western Blot: Protein hybridization • DNA sequencing: observe the DNA sequence • Solid-phase synthesis of nucleic acids: synthesize nucleic acids sequence de novo • PCR (polymerase chain reaction): amplify DNA billionfold

5. Overview Molecular Diagnostics and Therapeutics • Identify Changes in DNA Sequence • Identify Changes in Gene Expression • Individual Gene • Global Gene Expression Profiling • Develop Disease Targeted Therapies • Impact of the Human Genome Project

6. Human genome DNA Extraction • Venous blood(5 ml) → WBC → lysing buffer (SDS) and protease K → centrifugation, collect supernatant → phenol → centrifugation, collect supernant → RNase A and T1 → phenol → centrifugation, collect supernatant → potassium acetate and EtOH (absolute) → genomic DNA appears (like cotton fiber)

8. Restriction-enzyme analysis: “molecular scalpels”—Restriction endonucleases split DNA into specific fragment • RE : recognize specific base sequences in double-helical DNA and cleave at specific places. • Types of RE: >100

9. Nomenclature of RE: A 3-letter abbreviation for the host organism (italic!) + a strain designation + a roman numeral • E.g., EcoRI: (Escherichia coli) sticky end blunt end

10. Restriction fragments can be separated by gel electrophoresis and visualized • 1, 4,7: fX-174 RF DNA-Hind II digest; 2, 5, 8: lambda DNA-Hind III digest; 3, 6: fX-174 RF DNA-Hae III digest. Markers were separated for 30 min at 200 V in a 1% agarose gel, then stained with ethidium bromide(EB).

11. Electrophoresis

12. Horizontal electrophoresis system

13. Vertical electrophoresis system

14. Range of Separation in Cells Containing Different Amounts of Standard Low-EEO (electroendo-osmosis) Agarose: Agarose Concentration Range of Separation of in Gel (% [w/v]) Linear DNA Molecules (kb) 0.3 5 ~ 60 0.6 1 ~ 20 0.7 0.8 ~ 10 0.9 0.5 ~ 7 1.2 0.4 ~ 6 1.5 0.2 ~ 3 2.0 0.1 ~ 2

15. Effective Range of Separation of DNAs in Polyacrylamide gel (PAGE) Concentration of Effective Range of Separation Acrylamide Monomer (%) (bp) 3.5 1000 ~ 2000 5.0 80 ~ 500 8.0 60 ~ 400 12.0 40 ~ 200 15.0 25 ~ 150 20.0 6 ~ 100

16. Pulsed-field Gel Electrophoresis (PFGE) Resolution: 20 kb ~100 mb E.g., Gene Navigator Pulsed Field System (GE,USA)

18. Analyzing specific nucleic acids in complex mixtures • The Southern Blot- Edwin Southern • Digest the total DNA of an organism with a RE • fractionate by size • Identify sequence of interest using a labeled probe

19. Southern Blot:DNA hybridization

20. Southern Blot:DNA hybridization

21. Southern Blot:DNA hybridization

22. Southern blots are used for diagnostic procedures • Large deletions • Point mutations which alter a RE site • Chromosomal translocations • Gene amplification

23. Northern blot: RNA hybridization • Expression of a specific gene-changes from tissue to tissue • The relative size of the mRNA transcript • Relative levels of RNA in different samples

25. Northern Blot analysis reveals increased expression of β-globin mRNA in differentiated erythroleukemia cells. UN: uninduced cells

29. Western Blot:Protein hybridization

30. (A) Blot using the Dy4/6D3 Ab, which is specific for the dystrophin rod domian.(B) Blot using the Dy6/C5 Ab, which is specific for the C-terminal region of dystrophin.

31. DNA sequencing : Sanger (dideoxy) method, using flurescent tagged ddNTPs.

32. The 96-capillary 3730xl DNA Analyzer(ABI,USA) is the Gold Standard for high throughput genetic analysis

33. Frederick Sanger (1918-): Nobel Prize Winner 2 times: Pr.Sequencing/1958;DNA Sequencing/1980

34. DNA probes and genes can be synthesized by automated solid-phase methods: ABI 310 DNA Synthesizer

35. Millions of the target sequences can be readily obtained by PCR if the flanking sequences of the target are known. 3 steps: • Denature • Renature (annealing) • Extension ABI 9700 PCR System

37. PCR amplification of DNA Cycleds copies 3 2 4 4 5 8 10 256 20 262,144 30 268,435,456

38. PCR is used in many diagnostic tests and forensic tests • Point mutations can be detected by • incorporating them into the primers • PCR product can be sequenced • Sizing of repeated regions-microsatellite • Detecting infectious diseases (viral genomes)

39. PCR is powerful in lots of biomedical fields! Mullis K(1993 NP )

43. RT: Formation of a cDNA duplex

44. Quantitative RT-PCR Measures fluorescence generated by incorporation of a tagged nucleotide (SURF-4 mRNA)

45. The expression levels of thousands of genes can be simultaneously analyzed using DNA microarrays • The level at which a gene is expressed,as indicated by mRNA quantities,can vary widely,ranging from no expression to hundreds of mRNA copies per cell.Gene-expression patterns vary from cell type to cell type. • Even within the same cell, gene-expression levels may vary as the cell responds to changes in physiological circumstances.

46. DNA microarrays (gene chips)

48. Cells, cell lines, tumor specimens etc. Genes

49. Recombinant DNA technology • Recombinant DNA:Any DNA molecule formed in vitro by joining DNA fragments from different sources. Commonly produced by cutting DNA molecules with restriction enzymes and then joining the resulting fragments from different sources with DNA ligase.

50. Recombinant DNA technology: REs and DNA ligase (“molecular paste”) are key tools