19- microarray COVID19 PCR.ppt

Utilization of recombinant DNA as a diagnostic tool: Microarrays and PCR 1 * Learning objectives • What is microarray? • Principles of this technique. • The importance of the technique as a diagnostic tool. • The use of PCR in COVID 19 detection 2 * Background: • Major refinements of the techn...

Utilization of recombinant DNA as a diagnostic tool: Microarrays and PCR 1 * Learning objectives • What is microarray? • Principles of this technique. • The importance of the technique as a diagnostic tool. • The use of PCR in COVID 19 detection 2 * Background: • Major refinements of the technology underlying genomics, PCR, and nucleic acid hybridization have come together in the development of DNA microarrays (sometimes called DNA chips), which allow the rapid and simultaneous screening of many thousands of genes. • DNA microarrays (DNA chips) are small, solid supports on to which DNA samples corresponding to thousands of different genes are attached at known locations in a regular pattern of rows and columns. The supports themselves may be made of glass, plastic or nylon and are typically the size of a microscope slide. 3 How DNA microarray chip is made: The DNA samples, which may be gene-specific synthetic oligonucleotides (Photolithography) or cDNAs (RT-PCR), are spotted, printed, or actually synthesized directly on to the support phase. 4 * The types of microarray: • expression microarray: it is used to measure gene expression (mRNA) (slides 6). • comparative genomic microarray: in which the arrayed spots and the sample are both genomic DNA and can be used to detect loss or gain of genomic DNA that may be associated with certain genetic disorders. • mutation microarray: detects single nucleotide polymorphisms (SNPs) in the sample DNA of an individual. 5 Expression Microarray clip 6 Learning outcome Cy 3 Cy 5 7 PCR as a diagnostic tool in COVID 19 infection 8 COVID19 detection • RT-PCR technique was used to detect viral infection during COVID 19 pandemic since 2020. • It is used to transcribe and amplify specific SARSCoV-2 genomic sequence. A sample is taken from a suspected individuals and these sample are taken from different parts of the body such as nose (nasopharyngeal swabs) or throat (oropharyngeal swabs) by health care provider and is purified. it is treated with several chemical solution that remove substances such as proteins and fats and they extract only the viral genetic material RNA present in the sample. This will follow several steps to obtain the result of the sample either positive or negative (Next slide) 9 Procedure 10 • RT-PCR usually goes thirty-five cycles which means at the end of the process, 35 billion new copies of the targeted viral DNA are created from each strand of the virus which is available in the sample. As these copies are built, the TaqMan probe attached to the DNA strands will release a fluorescent dye which is measured by computer and presented in real time on the screen. the PC (computer) tracks the amount of the fluorescence in the sample after each cycle. • Cycle threshold (Ct value): when certain amount level of fluorescence dye is surpassed, this assure that the virus COVID-19 is there (present). The scientists also monitor the severity of the infection by measuring how many cycles it takes in the test. 11 • Cycle threshold value (Ct value) is agreed to be 35 cycles. Lower Ct values than 35 (indicating higher viral loads) means positive COVID19 case , while high Ct number than 35 (meaning less viral load) means negative COVID19 case. 12 Limitations • Possible corss contamination with other viral species • Gender effect • The quality and technique followed in the kit used • Sample intake = human factor 13