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DIAGNOSIS MOLEKULAR PENYAKIT

DIAGNOSIS MOLEKULAR PENYAKIT. Agustina Setiawati , MSc ., Apt. DIAGNOSIS MOLEKULAR PENYAKIT GENETIK. Agustina Setiawati , MSc ., Apt. OVALOSITOSIS. d elesi 27 bp. Tm = 4(G+C) + 2(A+T). Hasil PCR ovalositosis. Mana yang: sehat ? penderita ?. SICKLE CELL ANEMIA.

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DIAGNOSIS MOLEKULAR PENYAKIT

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  1. DIAGNOSIS MOLEKULARPENYAKIT AgustinaSetiawati, MSc., Apt

  2. DIAGNOSIS MOLEKULARPENYAKIT GENETIK AgustinaSetiawati, MSc., Apt

  3. OVALOSITOSIS delesi 27 bp

  4. Tm = 4(G+C) + 2(A+T)

  5. Hasil PCR ovalositosis Mana yang: sehat ? penderita ?

  6. SICKLE CELL ANEMIA

  7. GLUTAMAT VALIN • Kodonglutamat : GUU, GUC, GUA, GUG • Kodonvalin : GAA, GAG • PengenalanMstII: -CCTNAGG

  8. PemotonganenzimMstII

  9. PemotonganenzimCvnI

  10. THALLASEMIA • Mutasi pada gena globin sehingga jumlah/aktivitas produk menurun • Mutasi pada promoter – jumlah turun • Mutasi pada gena struktural – jumlah tetap aktivitas turun

  11. Thallesemia

  12. TIDAK SEMUA MUTASI PADA LOKUS RESTRIKSI • PCR/OLA • POLYMERASE CHAIN REACTION/ • OLIGONUCLEOTIDE LIGATION ASSAY • MUTASI PADA 106 A:T KE G:C • TARGET DIAMPLIFIKASI –PCR • HIBRIDISASI : PELACAK X DAN Y • LIGASI

  13. PCR/OLA • Like sickle cell anemia many genetic diseases are caused by mutant genes. • E.g.? • Many diseases are caused by a single nucleotide (nt) change in the wild type gene. • A single nt change can be detected by PCR/OLA ( oligonucleotide ligation assay). • E.g. The normal gene has A at nt position 106 and mutant has a G. • 2 short oligonucleotides (oligo) are synthesized • Oligo 1 (probe x) is complementary to the wild type has A at 106 (3’ end).

  14. PCR/OLA • Oligo 2 ( probe y) has G at 107 (5’ end). • The two probes are incubated with the PCR amplified target DNA. • For the wild type the two probes anneal so that the 3’end of probe x is next to the 5’end of probe y. • For the mutant gene the nt at the 3’ end of probe x is a mismatch and does not anneal.

  15. PCR/OLA • DNA ligase is added. The two probes will only ligate if the two probes are perfectly aligned (as in the wild type). • To determine if the mutant or wild type gene is present it is necessary to detect for ligation. • Probe x is labeled at 5’ end with biotin • Probe x is labeled at 5’ end with digoxygenin.

  16. PCR/OLA • Digoxygenin serves as an antibody binding indicator. • After washing a colourless substrate is added. • If a coloured substrate appears this is indicative that the biotin probe (x) ligated to the dioxygenin probe (Y) and that the wild type gene is present.

  17. PCR/OLA

  18. PCR/OLA

  19. DETEKSI MUTASI SATU BASA DENGAN PCR

  20. DETEKSI MUTASI DI BB TEMPAT • PCR • HIBRIDISASI • PELACAK 1, 2, 3, 4, 5, 6, 7, 8 PADA MEMBRAN • PCR BGN TARGET TERMUTASI+BIOTIN • NORMAL (-), MUTAN (+) • HIBRIDISASI

  21. Analisis SSCP, mobility shift

  22. Enzymes as Therapeutic Agents/ DNase1 • Cystic fibrosis (CF) is one of the most fatal heredity diseases among European and their descendants with ~30,000 cases in the US and 23,000 in Canada. • Furthermore among European descendants it occurs in 1 in 2,500 live birth and 1 in 25 are carriers. • It is caused by more than 500 different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. • Individuals with CF are highly susceptible to bacterial infection and antibiotic treatment often results in resistant strains.

  23. DNase 1 • A thick mucus which is a results of: • Alignate produced by bacteria • DNA from lysed cells • Leucocytes which accumulate due to the infection • Makes breathing difficult. • Scientist at Genentech isolated the gene for DNase1 • The purified enzyme was delivered as an aerosol to the lung where it hydrolysed the DNA into short oligionucleotides. • This decrease the viscosity in the lungs and made breathing easier.

  24. Alginate Lyase • Alginate is a polysaccharide polymer that is produced by seaweed and some soil and marine bacteria. • The excretion of alginate by Pseudomonas aeruginosa of patients with CF contributes to the viscosity in the lung. • The enzyme alginate lyase can liquefy bacteria alginate. • Alginate lyase was isolate from Flavobacteriumsp. and cloned into E. coli.

  25. Aliginate Lyse • The expressed gene produced a protein of 69,000 Da. • The 69,000 Da protein produced a proteolytic enzyme of 6,000 Da. • The remain 63,000 Da protein was cleaved to produce a 43,000 Da which is able to liquefy bacterial alginate. • Combined with DNase1, alginate lyse is able to reduce the mucus in the lungs of patients with CF.

  26. DNAse 1 and Alginate lyase

  27. CYTIC FIBROSIS • Delesi satu asam amino fenilalanin pada kodon 508 CFTR (Cytic Fibrosis Transmembrane Regulator) • Bagaimana cara mendeteksinya

  28. Deteksi fusi gena - leukemia • Translokasi kromosom 9 dan 22 pada q34 dan q11 • Translokasi kromosom 11 dam 17 pada q 22 dan q21 • Bagimana cara mendeteksinya ?

  29. Gleevec for chronic myeloid leukaemia (CML) • .

  30. TODAY Deteksi Molekuler Penyakit Genetik TERIMA KASIH

  31. ENZYME BASED • ANTIBODY BASED • DNA BASED DIAGNOSIS MOLEKULARPENYAKIT INFEKSI AgustinaSetiawati, MSc., Apt

  32. Problems? Traditionally diagnosis of infection based on finding parasite • some parasites morphologically indistinguishable • parasites hidden in various host tissue

  33. Skin

  34. Traditional diagnosis of Malaria

  35. Lumbar Puncture for Sleeping sickness

  36. THE SOLUTION ? Current laboratory techniques not entirely satisfactory Need trained staff, equipment, slow throughput Rapid molecular tests being developed

  37. ENZYME BASED (-) (+) • simple technique. • large number of typing enzymes available • many samples typed at same time • power to distinguish morphologically similar parasites. • Significant tissue needed for analysis visceral leishmaniasis requires spleen, liver, • Technique not rapid  can take days • Sometimes incorrect diagnosis  enzyme labile • Technique simple but equipment • expensive

  38. Iso-enzymes separated by charge: Isoelectric focusing equipment

  39. Enzymes separated by size: SDS-PAGE

  40. ANTIBODY BASED (-) (+) • rapid easy field based tests can be developed • useful for both individual & mass population screening • cannot distinguish past/ present infections • cannot distinguish morphologically similar parasites • expensive to develop • significant research prior to commercialization

  41. Enzyme-Linked Immunosorbant Assay (ELISA) Positive Negative

  42. DNA BASED • Nonculturable agents • Human papilloma virus • Hepatitis B virus • Fastidious, slow-growing agents • Mycobacterium tuberculosis • Legionellapneumophilia • Highly infectious agents that are dangerous to culture • Francisellatularensis • Brucella species • Coccidioidisimmitis

  43. DNA BASED • In situ detection of infectious agents • Helicobacter pylori • Toxoplasma gondii • Agents present in low numbers • HIV in antibody negative patients • CMV in transplanted organs • Organisms present in small volume specimens • Intra-ocular fluid • Forensic samples

  44. DNA BASED • Restriction endonuclease analysis • PCR • DNA Hybridization • DNA fingerprinting

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