DIAGNOSIS MOLEKULAR
PENYAKIT
Agustina Setiawati, MSc., Apt
DIAGNOSIS MOLEKULAR
PENYAKIT GENETIK
Agustina Setiawati, MSc., Apt
OVALOSITOSIS
delesi 27 bp
Tm = 4(G+C) + 2(A+T)
Mana yang:
sehat ?
penderita ?
GLUTAMAT
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VALIN
Kodon glutamat : GUU, GUC, GUA, GUG
Kodon valin : GAA, GAG
Pengenalan MstII: -CCTNAGG
Pemotongan enzim MstII
Pemotongan enzim CvnI
THALLASEMIA
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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
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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
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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
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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
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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.
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PCR/OLA
PCR/OLA
DETEKSI MUTASI SATU BASA DENGAN PCR
DETEKSI MUTASI
DI BB TEMPAT
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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
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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
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A thick mucus which is a results of:
Alignate produced by bacteria
 DNA from lysed cells
 Leucocytes which accumulate due to the infection
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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
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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
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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
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Delesi satu asam amino fenilalanin pada kodon
508 CFTR (Cytic Fibrosis Transmembrane
Regulator)
Bagaimana cara mendeteksinya
Deteksi fusi gena - leukemia
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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)
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.
TODAY
Deteksi
Molekuler
Penyakit
Genetik
TERIMA KASIH
DIAGNOSIS MOLEKULAR
PENYAKIT INFEKSI
Agustina Setiawati, MSc., Apt
Problems?
Traditionally diagnosis of infection based on
finding parasite
some parasites morphologically
indistinguishable
 parasites hidden in various host
tissue
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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
(+)
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simple technique.
large number of typing
enzymes
available
many samples typed at
same time
power to distinguish
morphologically
similar parasites.
(-)
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Significant tissue needed
for analysis
 visceral leishmaniasis
requires spleen, liver,
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Technique not rapid
 can take days
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Sometimes incorrect
diagnosis
 enzyme labile
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Technique simple but
equipment
expensive
Iso-enzymes separated by charge:
Isoelectric focusing equipment
Enzymes separated by size: SDS-PAGE
ANTIBODY BASED
(+)
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rapid easy field based
tests can be
developed
useful for both
individual & mass
population screening
(-)
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cannot distinguish
past/ present infections
cannot distinguish
morphologically
similar parasites
expensive to develop
significant
research prior to
commercialization
Enzyme-Linked Immunosorbant Assay
(ELISA)
Positive
Negative
DNA BASED
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Nonculturable agents
Human papilloma virus
 Hepatitis B virus
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Fastidious, slow-growing agents
Mycobacterium tuberculosis
 Legionella pneumophilia
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Highly infectious agents that are dangerous to
culture
Francisella tularensis
 Brucella species
 Coccidioidis immitis
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DNA BASED
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In situ detection of infectious agents
Helicobacter pylori
 Toxoplasma gondii
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Agents present in low numbers
HIV in antibody negative patients
 CMV in transplanted organs
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Organisms present in small volume specimens
Intra-ocular fluid
 Forensic samples
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DNA BASED
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Restriction endonuclease analysis
PCR
DNA Hybridization
DNA fingerprinting
DNA FINGERPRINT
Any question?