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Early 90’s:

Molecular biology approach used as a diagnostic tool

Genetic diseases

Infectious diseases

Molecular diagnosis

Molecular Diagnosis In Genetic Diseases

Differential diagnosis 2. Carrier detection in the family 3. Presymptomatic diagnosis . Carrier detection in the population 4. Prenatal diagnosis 5.Application: 1.

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 .

• penentuan chromosome sex prenatal.Time limitation: (Examples) Congenital Adrenal Hyperplasia • 21-hydroxylase deficiency Reduced cortisol production.salt losing • deteksi mutasi pada neonatus.virilization . . .

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 .




Sickle-cell disease .



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 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 .

Young Churchill Livingstone Molecular Diagnosis of Genetic Diseases Rob Elles Human Press . Ian D.Emery’s Elements of Medical Genetics Robert F. Mueller.


and generate a test sample sufficient to detect the presence or absence of a specific virus. even from just one cell. copy its genetic sequence over and over.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.bacterium or any particular sequence of genetic material .

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

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


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


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.They are specific for the target region of the organism . with labeled 5’ end to aid in detection. synthetic sequences of singlestranded DNA typically consisting of 20 – 30 bases.

This allows the primers to anneal to the target sequence with high specificity . 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.TWO PRIMERS • 2 PRIMERS are included in the PCR. depending on the length and bases sequence of the primers. one for each of the complimentary single DNA strands that was produced during denaturation.

PCR Step 3 Taq DNA polymerase catalyses Primer Extension as complimentary nucleotides are incorporated .

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.EXTENSION • Once the primers anneal to the complimentary DNA sequences.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) .

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

PCR Step 4 End of the first PCR cycle:results in 2 copies of target sequence .

PCR M. line 5 M. line 6 M.tuberculosis show the sensitivity up to 10 pg DNA Line 1 : marker phageX174HaeIII. line 2: M.tbc 1ng.smegmatis as negative control.tbc 50pg.tbc as positive control 10 ng line 3 M.tbc 5ng. line 7 M.tbc 10 pg .tbc 500 pg. line 8 M. line 4 M.

tbc .Result of PCR to detect existence of M.


as such.PCR and infectious disease • PCR technology facilitates the detection of DNA or RNA of pathogenic organisms. as patients can takes weeks to develop antibodies against infectious agent . 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.

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

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

helping to narrow the window period .HCV. in blood. namely viral DNA or RNA.HBV.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.

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. such as heart disease or cancer . including Cystic fibrosis • In the future may be used in predictive testsmethods for finding out who is predisposed to common disorders.

• Can not be used for single diagnostic. as an existing microbes for example.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. 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 .

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

Nanopores reading the genetic code g e n e .

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 .Nanosphere Assays Nanosphere’s detection hardware Spot Assay A B Genotyping SNP’s in PCR samples C 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.

Microchip proteins Array-I .

Microchips Arrays-II .

Isolation and Sequencing a Specific Peptides .

Data and Result .

Results .