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DIAGNOSIS MOLEKULARPENYAKIT
Agustina Setiawati, MSc., Apt
Agustina Setiawati, MSc., Apt
DIAGNOSIS MOLEKULARPENYAKIT GENETIK
OVALOSITOSIS
delesi 27 bp
Tm = 4(G+C) + 2(A+T)
Mana yang:sehat ?penderita ?
GLUTAMAT VALIN
Kodon glutamat : GUU, GUC, GUA, GUG Kodon valin : GAA, GAG Pengenalan MstII: -CCTNAGG
Pemotongan enzim MstII
Pemotongan enzim CvnI
THALLASEMIA Mutasi pada gena globin sehingga
jumlah/aktivitas produk menurun Mutasi pada promoter – jumlah turun Mutasi pada gena struktural – jumlah
tetap aktivitas turun
Thallesemia
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
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).
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.
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.
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.
PCR/OLA
PCR/OLA
DETEKSI MUTASI SATU BASA DENGAN PCR
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
Analisis SSCP, mobility shift
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.
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.
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.
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.
DNAse 1 and Alginate lyase
CYTIC FIBROSIS Delesi satu asam amino fenilalanin pada
kodon 508 CFTR (Cytic Fibrosis Transmembrane Regulator)
Bagaimana cara mendeteksinya
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 ?
Gleevec for chronic myeloid leukaemia (CML)
.
TODAYDeteksi MolekulerPenyakit Genetik
TERIMA KASIH
Agustina Setiawati, MSc., Apt
DIAGNOSIS MOLEKULARPENYAKIT INFEKSI
ENZYME BASED ANTIBODY BASED DNA BASED
Problems?Traditionally diagnosis of infection based on finding parasite some parasites morphologically indistinguishable parasites hidden in various host tissue
Skin
Traditional diagnosis of Malaria
Lumbar Puncture for Sleeping sickness
THE SOLUTION ?
Current laboratory techniques not entirely satisfactory Need trained staff, equipment, slow throughput
Rapid molecular tests being developed
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
(+) (-)
Iso-enzymes separated by charge: Isoelectric focusing
equipment
Enzymes separated by size: SDS-PAGE
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
Enzyme-Linked Immunosorbant Assay (ELISA)
Positive Negative
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
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
Restriction endonuclease analysis PCR DNA Hybridization DNA fingerprinting
DNA BASED
DNA FINGERPRINT
Any question?