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MOLECULAR DIAGNOSIS

Early 90s:

Molecular biology approach used as a diagnostic tool

Genetic diseases

Infectious diseases

Molecular diagnosis

Molecular Diagnosis In Genetic Diseases

Application:
1. Differential diagnosis 2. Carrier detection in the family 3. Carrier detection in the population

4. Prenatal diagnosis
5. Presymptomatic diagnosis

Differential diagnostic testing X-linked muscular dystrophies


Other neurological disorders Fragile X disease Other cause of mental retardation

Carrier detection within family Congenital adrenal Hyperplasia

Carrier detection within population For autosomal recessive disorders


Cystic Fibrosis Not efficient for hemoglobinopathies

Prenatal Diagnosis
Severe childhood onset diseases with a poor prognosis and no effective treatment

Presymptomatic diagnosis
For adult onset disorders

Time limitation: (Examples)


a-thalassemia
Carrier detection in pregnancy

Prenatal diagnosis

Time limitation: (Examples)


Congenital Adrenal Hyperplasia
21-hydroxylase deficiency Reduced cortisol production; - virilization - salt losing
deteksi mutasi pada neonatus. penentuan chromosome sex prenatal.

Mutation detection:
Specimens: peripheral blood/cord blood lymphocytes villi choriales amniotic fluid Methods: PCR PCR-RFLP (restriction fragment length polymorphism) oligo specific probe ARMS (amplification refractory mutation system) etc

PCR

Sickle-cell disease

PHENYLKETONURIA (PKU)
Inborn errors of metabolism

Mutation in gene encoding phenylalanine hydroxylase Phenylalanine Tyrosine


A cause of mental retardation --- can be corrected

Newborn screening assay of blood phenylalanine levels

ANTIGEN DETECTION

Antigen is a gene product Antigen detection is not a molecular diagnostic

Antibody used as a detector: polyclonal antibodies recognized several epitopes monoclonal antibody recognized one epitope

Emerys Elements of Medical Genetics


Robert F. Mueller, Ian D. Young Churchill Livingstone

Molecular Diagnosis of Genetic Diseases


Rob Elles Human Press

PCR BASED DIAGNOSTIC IN MICROBIOLOGY, VIROLOGY AND INFECTIOUS DISEASE

Application of PCR
PCR technology has become an essential research and diagnostic tool for improving human health and quality of life

PCR technology allows scientists to take a specimen of genetic material, even from just one cell, copy its genetic sequence over and over, and generate a test sample sufficient to detect the presence or absence of a specific virus,bacterium or any particular sequence of genetic material

Benefit
Medical research and clinical medicine are benefiting from PCR technology mainly in two areas : - detection of infectious organisms, including AIDS and hepatitis virus, atypic bacteria such as chlamydia, and TBC - detection of genetic variations,including mutations, in human genes

The PCR Process


PCR is closely patterned after the natural principle of DNA replication One PCR cycle consists of the following steps : - Denaturation - Annealing - Extension This process takes place in a thermal cycler, an instrument that automatically controls and alternates the temperatures for programmed period of time for the appropriate number of PCR cycles (30 40 cycles)

PCR step 1

DENATURATION BY HEAT

Denaturation by heat
Heat usually > 90 degree C, separates double stranded DNA into two single strands, referred to as Denaturation The hydrogen bond linking the bases to one another are weak, they break at high temp., whereas the bonds between deoxyribose and phosphates,which are stronger covalent bonds, remain intact

PCR step 2 ANNEALING

Annealing Primer binding to the target


The goal is not to replicate the entire strand of DNA but to replicate a target sequence of approximately 100-600 bp that is a unique to the organism PRIMERS mark the ends of the target sequence. Test primers =AMPLICOR are short, synthetic sequences of singlestranded DNA typically consisting of 20 30 bases, with labeled 5 end to aid in detection.They are specific for the target region of the organism

TWO PRIMERS
2 PRIMERS are included in the PCR, one for each of the complimentary single DNA strands that was produced during denaturation. The beginning of the DNA target sequence of interest is marked by the primers that anneal (bind) to the complimentary sequence Annealing temp : 40 65 degree C, depending on the length and bases sequence of the primers. This allows the primers to anneal to the target sequence with high specificity

PCR Step 3

Taq DNA polymerase catalyses Primer Extension as complimentary nucleotides are incorporated

EXTENSION
Once the primers anneal to the complimentary DNA sequences, the temperature is raised to approximately 72 degree C and the enzyme Taq DNA polymerase is used to replicate the DNA strands Taq DNA polymerase,begins the synthesis process at the region marked by the primers. It synthesizes new double stranded DNA mol., both identical to the original stranded target DNA region, by facilitating the bindings and joining of the complementary nucleotides that are free in solution (dNTPs)

EXTENSION
Extension always begins at the 3 end of the primer making a double strand out of each of the two single strands. Taq DNA polymerase synthesizes exclusively in the 5 to 3 direction. Therefore, free nucleotides in the solution are only added to the 3 end of the primers constructing the complimentary strand of the targeted DNA sequence

PCR Step 4

End of the first PCR cycle:results in 2 copies of target sequence

PCR M.tuberculosis show the sensitivity up to 10 pg DNA

Line 1 : marker phageX174HaeIII, line 2: M.tbc as positive control 10 ng line 3 M.tbc 5ng, line 4 M.smegmatis as negative control, line 5 M.tbc 1ng, line 6 M.tbc 500 pg, line 7 M.tbc 50pg, line 8 M.tbc 10 pg

Result of PCR to detect existence of M.tbc

PCR and infectious disease


PCR technology facilitates the detection of DNA or RNA of pathogenic organisms, as such, is the basis for a broad range of clinical diagnostic test for various infectious agents,including viruses and bacteria It is antigen detection method PCR-based test are able to detect the presence of pathogenic agents earlier than serologically-based methods, as patients can takes weeks to develop antibodies against infectious agent

Quantitative PCR
PCR-based tests is also developed, and now designed to quantify the amount of virus in a persons blood = VIRAL LOAD Viral load allow physicians to monitor their patients disease progression and response to therapy. Viral load assessment before, during, and after therapy has tremendous potential for improving the clinical management of diseases caused by viral infection,I.e.AIDS, hepatitis

RELIABLE AND GOOD PCR DIAGNOSTIC


PCR-based diagnostics tests available in the market for detecting and / or quantifying several pathogens : - HIV-1 which causes AIDS - Hepatitis B and C which can lead to liver Ca - Human Papilloma Virus which can lead to infertility in women - Neisseria gonnorhoea, especially for women - Cytomegalovirus - Mycobacterium tuberculosis

PCR and Blood Screening


.A small risk of viral transmission remains primarily due to the failure of a screening tests to identify recently infected donor during the window period Test using PCR nucleic acid amplification testing technology detect the actual virus Highly sensitive PCR-based tests are available for detecting early markers of HIV-1,HBV,HCV, namely viral DNA or RNA, in blood, helping to narrow the window period

PCR and genetic testing


PCR technology can be used to easily distinguish among the tiny variations in DNA that make people genetically unique Available PCR test for genetic disorders, including Cystic fibrosis In the future may be used in predictive testsmethods for finding out who is predisposed to common disorders, such as heart disease or cancer

Weakness
Should be done by highly skilled person Appropriate specimen sometimes not easy The service lab, must be designed as such to minimize contamination with other genetic materials. Can not be used for single diagnostic, as an existing microbes for example, maybe not the cause of the expressed disease syndrome

What is nanotechnology?

So small 1/80.000 of human hair diameter Can be used for Diagnosis, treatment and im cancer detection

DNA microarray

Quantum dots can find cancer signature

Variations causing harmful changes


There are a group of variations in coding and regulatory regions that result in harmful effects. These are called mutations. They cause disease because changes in the genome's instructions alter the functions of important proteins that are needed for health. For example, diabetes, cancer, heart disease, Huntington's disease, and hemophilia all result from variations that cause harmful effects. In a "simple" disease such as hemophilia, variation in one gene is sufficient to cause disease symptoms. By contrast, in a "complex" disease like cancer, symptoms are seen only after many variations have occurred in different genes in the same cell.

Nanopores reading the genetic code


g e n e

Nanosphere Assays
Nanospheres detection hardware
Spot Assay

Genotyping SNPs in PCR samples

Genomic DNA detection

A: Specific nanoprticle probes allow to do identification and differentiation of single nucleotide poly [SNP]-Nanosphere spot assay format provides DNA sequence information without any fluorescen Chemiluminescent, radioactive or electrochemical methods. B: SNP from the factor V leiden gene associated with hypercoagulation C: DNA Genomic detection without PCR amplification of the MTHFR gene [associated with hypercoa

Microchip proteins Array-I

Microchips Arrays-II

Isolation and Sequencing a Specific Peptides

Data and Result

Results

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