DIAGNOSIS MOLEKULAR PENYAKIT Agustina Setiawati, MSc., Apt.

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1 DIAGNOSIS MOLEKULAR PENYAKIT Agustina Setiawati, MSc., Apt

2 DIAGNOSIS MOLEKULAR PENYAKIT GENETIK

3 OVALOSITOSIS delesi 27 bp

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

5 Mana yang: sehat ? penderita ?

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9 GLUTAMAT VALIN  Kodon glutamat : GUU, GUC, GUA, GUG  Kodon valin : GAA, GAG  Pengenalan MstII: -CCTNAGG

10 Pemotongan enzim MstII

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12 Pemotongan enzim CvnI

13 THALLASEMIA  Mutasi pada gena globin sehingga jumlah/aktivitas produk menurun  Mutasi pada promoter – jumlah turun  Mutasi pada gena struktural – jumlah tetap aktivitas turun

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15 Thallesemia

16 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

17 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).

18 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.

19 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.

20 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.

21 PCR/OLA

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23 DETEKSI MUTASI SATU BASA DENGAN PCR

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25 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

26 Analisis SSCP, mobility shift

27 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.

28 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.

29 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 Flavobacterium sp. and cloned into E. coli.

30 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.

31 DNAse 1 and Alginate lyase

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

33 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 ?

34 Gleevec for chronic myeloid leukaemia (CML) ..

35 TODAY Deteksi Molekuler Penyakit Genetik TERIMA KASIH

36 Agustina Setiawati, MSc., Apt DIAGNOSIS MOLEKULAR PENYAKIT INFEKSI

37 Problems? Traditionally diagnosis of infection based on finding parasite  some parasites morphologically indistinguishable  parasites hidden in various host tissue

38 Skin

39 Traditional diagnosis of Malaria

40 Lumbar Puncture for Sleeping sickness

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

42 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 (+) (-)

43 Iso-enzymes separated by charge: Isoelectric focusing equipment

44 Enzymes separated by size: SDS-PAGE

45  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 (+) (-) ANTIBODY BASED

46 Enzyme-Linked Immunosorbant Assay (ELISA) Positive Negative

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48 DNA BASED  Nonculturable agents  Human papilloma virus  Hepatitis B virus  Fastidious, slow-growing agents  Mycobacterium tuberculosis  Legionella pneumophilia  Highly infectious agents that are dangerous to culture  Francisella tularensis  Brucella species  Coccidioidis immitis

49 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

50 RRestriction endonuclease analysis PPCR DDNA Hybridization DDNA fingerprinting DNA BASED

51 DNA FINGERPRINT

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54 Any question?