5 Add chemiluminescent substrate 6 Put membrane in the UVP machine to detect

5 add chemiluminescent substrate 6 put membrane in

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5. Add chemiluminescent substrate 6. Put membrane in the UVP machine to detect Checkboard DNA-DNA hybridization: A high throughout version of dot blot used for analysis of dental samples: detection and semi-quantification of up to 40 bacterial species in up to 28 dental samples using whole genomic probes. Fluorescent In situ hybridization: (FISH) Uses a DNA or RNA probe, usually fluorescent, to detect and localize a DNA or mRNA in a tissue or cells e.g. abnormal chromosome, gene mapping, toxicological studies, bacteria. Cells are arrested at metaphase Steps: 1. Deparaffinized slide in xylene 3 times at room T for 5 minutes each 2. Dehydrate slide in 100% ethanol twice for 5 min each. 3. Pre-heat paraffin pretreatment sol. to 95C make tissue permeable to probe 4. Immerse slide in pre-heated paraffin for 30 min 5. Immerse slide in 2xSSC twice for 5 min each
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6. Protease treatment: pre-heat protease to 37C digest protein on the surface, so more probe can go in. 7. Dehydrate with ethanol again 8. Air dry 9. Add FISH probe, cover w cover glass, seal w rubber cement 10. Denature by heating the slide at 75 C. 11. Hybridization by putting the slide in humidified box, remove rubber cement, remove cover glass. 12. Immersed slide in pre-heaated 2xSSC 13. Add DAPI is dye for any DNA, binds strongly to A-T region in DNA. DNA microarrays: Definition: a glass or silicon of chip with thousands of unlabeled DNA probes spotted on it for hybridization with labeled nucleic acids from samples. Each spot of DNA, called a probe, represents a single gene. Uses: detection and quantification of microbes in a sample study/compare gene expression detect gene mutation (SNPs) detect gene copy variations (competitive genomic hybridization) Steps: 1. Sample preparation 2. Purification : aqueous phase contains mRNA, phenol phase contains protein and DNA. 3. Reverse trx.: mRNA to cDNA 4. Fluorescently labeled with Cy3 (green) and Cy5 (red) 4. Combine equal amount and hybridize on microarray. 5. Scanning Green: express in more healthy tissues Yellow: both equally expressed in both tissues Red: more expressed in disease tissues Questions: 1. which technique would u use to detect gene expression from different individuals? 2. which of the following tools of genetic engineering is used to make amplify or make multiple copies of a small sample of DNA? PCR 3. Labeled, known, short stretches of DNA used to detect a specific of nucleotides in a mixture known as gene probes
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Nucleic Acids Amplification techniques What are they? Techniques that involve making identical copies of a specific DNA segment in detectable quantities in vitro. Uses: Pathogen detection/quantification: Detection of resistance and virulence genes Genotyping Gene expression Mutations/allelic discrimination Preparing libraries for sequencing Methods: Thermal-cycling-dependent a) Polymerase chain reaction (PCR) b) Ligase chain rxn (LCR) Isothermal a) Nucleic acid sequence-based amplification (NASBA) b)
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  • Summer '17
  • Al-Hebshi
  • DNA, RNA

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