diagnosis molekular penyakit

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?