Professional Documents
Culture Documents
Early 90s:
Genetic diseases
Infectious diseases
Molecular diagnosis
Application:
1. Differential diagnosis 2. Carrier detection in the family 3. Carrier detection in the population
4. Prenatal diagnosis
5. Presymptomatic diagnosis
Prenatal Diagnosis
Severe childhood onset diseases with a poor prognosis and no effective treatment
Presymptomatic diagnosis
For adult onset disorders
Prenatal diagnosis
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
ANTIGEN DETECTION
Antibody used as a detector: polyclonal antibodies recognized several epitopes monoclonal antibody recognized one epitope
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
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
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
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
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
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
Nanosphere Assays
Nanospheres detection hardware
Spot Assay
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
Microchips Arrays-II
Results