Microarray and PCR Diagnostic Tool PDF

* Learning objectives Utilization of recombinant DNA as a diagnostic tool: Microarrays and PCR • What is microarray? • Principles of this technique. • The importance of the technique as a diagnostic tool. • The use of PCR in COVID 19 detection 1 2 How DNA microarray chip is made: - * Backgroun...

* Learning objectives Utilization of recombinant DNA as a diagnostic tool: Microarrays and PCR • What is microarray? • Principles of this technique. • The importance of the technique as a diagnostic tool. • The use of PCR in COVID 19 detection 1 2 How DNA microarray chip is made: - * 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. 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 3 - Photolithography a chemical synthesis of oligonucleotides, where a light source is used to create the oligonucleotides. Note: - Genomics everything about the DNA (structure, properties, replication and so on) - PCR (polymerase chain reaction) of interest Note: - cDNAs (RT-PCR) and Photolithography to create the DNA chips these are ways amplification of the gene - nucleic acid hybridization the way of interaction between oligonucleotides (for example probes used in gene cloning: hybridization of probes with gene of interest) - in the sequencing unit, the oligonucleotides strands are single strand sequence. - The DNA chips come in a kit from the company, the box contain (the microchip with barcode, and manual), the manual have the name of gene in each spot. - microarray is also called Affymetrix. - The microchip which is in the market contain a whole genome of bacterial cell or viral cell, or a whole genome of plant cells, but rarely there is microchip with whole human genome because humans have huge number of genes. Note: * 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. - comparative genomic microarray there is a genome of a microorganism, for example scientists discovered a new bacterium and they want to compare it with other bacterial families to see what the similarities are (so in this case use a microchip with genome of the known bacteria and compare with the genome of newly discovered bacteria), it is mostly used to compare species. 5 Expression Microarray clip: http://learn.genetics.utah.edu/content/labs/microarray/ Note: 1- There are healthy and cancer cells samples (one is the sample healthy cells and the other one you have doubt about and want to compare them with the standard cells) 2- One if the major features of cancer cells is the uncontrolled multiplications due to the genetic mutation. Also sometimes, they over express some genes like more mRNA than normal cells Steps to do microarray: 3- Collect tissue: Take a sample from the defected organ and from a normal skin tissue 4- Isolate RNA: in this step we want to extract the RNA from the cell and separate the DNA, proteins and other cell components leaving the RNA in the solution • • • • Put a solvent in the sample tissue and the healthy tissue Use a (Vortex-Genie) to mix the sample and the tissue will dissolve and RNA will be released Use centrifuge to separate the RNA from the rest of cell contents the DNA, proteins and other stuff will precipitate because they are larger, and the RNA will be in the supernatant. Take the supernatant having the RNA from the healthy tissue tube and the cancer cell tube using a micropipette and put it in a new tube, then move to next step. 5- Isolate mRNA: • in the tube from previous step, we have mRNA, tRNA, rRNA and in microarray our goal is mRNA • one of the eukaryotic mRNA features is the poly-A tail. • in this step we use affinity chromatography, we use columns filled with small beads having (poly-T), so that it will complement the poly-A in the mRNA. • Use a buffer to wash the columns and detach mRNA from the poly-T beads (it will change the chemical conditions thus it will not allow hyperidization and thus the mRNA will detach from the poly-T beads) 6- Make labeled DNA copy: (dye and do RT-PCR) • Use cyanin dyes: CY 3 (green), CY5 (red) these dyes are connected to nucleotides • Add the green dye with healthy mRNA then use RT-PCR, the reverse transcriptase will use the dyed nucleotides to create cDNA (green) this will be further amplified by the PCR. • Add the red dye with cancer mRNA then use RT-PCR, the reverse transcriptase will use the dyed nucleotides to create cDNA (red) this will be further amplified by the PCR. 7- Apply DNA: • The tubes now have cDNA (double strands) and in the microarray there are fixed single stranded DNA sequence, so before we add them to the microarray, we must denature the DNA in the tubes to have single strands so that they can interact with the single strands in the microarray. • The database in the computer has a list of which gene is contained in each spot. • Add the sample from the healthy then from the cancer or (suspected sample), so there will be a competition between the denatured cDNA from the healthy and suspected sample for hyperidization with the ssDNA in the microchip • Wash off the excess cDNA that didn’t bind 8- Scan Microarray: use a scanner to analyze the data and show the colored spots in the computer 9- Analyze the Data: o Green spot o Red spot represent damage or overexpression if this gene is associated with a type of cancer o Yellow or orange spot o Black spot normal cell if we get this, we go check in the manual to see the gene type and the gene expressed in both normal and suspected the gene in this spot is not expressed in both cells Learning outcome 7 COVID19 detection PCR as a diagnostic tool in COVID 19 infection • RT-PCR technique was used to detect viral infection during COVID 19 pandemic since 2020. • It is used to transcribe and amplify specific SARS-CoV-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) 8 Note: - Initially it was not designed for diagnostic purpose. It was used for research only. 9 - There are two ways to detect COVID19 through blood (detecting antigen and antibodies), through PCR - The blood (detecting antigen and antibodies) method is not very sensitive and not very effective and using a type of PCR was much better. - They used RT-RCR Note: - For sampling swap preferred to take both nose and throat which uses a uniquely made probe Note: - First convert the take sample Procedure - Isolate the RNA - Convert RNA to cDNA - 10 - Amplify and use a probe called (TaqMan probe), to detect how much genome of the virus in the patient sample the probe has a fluorescent dye (Reporter dye) and a Quencher, this probe is very selective to a reserved sequence in the corona virus gene, the probe will attach to the sequence first then the polymerase will degrade the probe, this will lead to release of the dye and quencher, this will give a signal (before the cleavage the dye was masked by the quencher), if there is a signal then there is genome of the virus. • 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. • 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 12 For example, if Ct value is 20, this patient is positive, if the signal comes after Ct 36 or 37, 38.. the patient is negative. severity of patient having Ct 20 is more (has higher viral loads) than someone with Ct value 30 Limitations: - Sample intake and handling - Virus mutations this is a challenge for the probe because it is designed for the original corona virus, in case if mutation they need to make a new probe - Contamination in the sample (other viral particles of similar viral families) - Age and gender - Quality of the kits used and the different standard