Nucleic Acid Measurements - 1302 Words

Lecture 8 Nucleic Acid-Based Measurements Text Chapter 13 Wednesday, July 24, 2013 Total community DNA †¢ Extract DNA from soil – – – – remove cells from soil separate cells from soil lyse cells separate DNA from cells – purify DNA †¢ Extract DNA from soil – Extract DNA from cells in presence of soil †¢ Bead-beating †¢ chemical or enzymatic treatment – Sodium dodecyl sulfate or lysozyme Wednesday, July 24, 2013 DNA purification †¢ Cesium chloride gradient centrifugation †¢ Kits Low density DNA High density Wednesday, July 24, 2013 Visualizing nucleic acidsBlotting †¢ Southern blotting – DNA †¢ Northern blotting – RNA Wednesday, July 24, 2013 Agarose gel electrophoresis - Stain gel with ethidium†¦show more content†¦ednesday, July 24, 2013 50  µm Combining ELF and CF319a probes to determine what portion of the PO4aseactive cells in floc fall within cytophaga-flavobacteria group ELFTM PO4ase CF319a FISH probe activity FISH probe PO4ase activity 17% of total community PO4ase activity contributed by cytophaga Wednesday, July 24, 2013 Summary FISH provides information on – Presence of specific populations – Morphology of specific populations – Relative numerical contribution of specific populations to total community – Spatial relationships between populations – Functions associated with specific populations Wednesday, July 24, 2013 Gene probe detection of a DNA sequence Digoxigenin (DIG) Probes range in size from 18-100bp ssDNA Denatured ssDNA from suspect bacterium Wednesday, July 24, 2013 Alternative gene probe detection of a DNA sequence Wednesday, July 24, 2013 Exploring microbial activity †¢Need to know sequence of gene being expressed †¢Alternatively, use random hexamer primers, then sequence cDNA product to identify gene being expressed Wednesday, July 24, 2013 Reverse transcriptase PCR (RT-PCR) †¢ Make single-strand cDNA from mRNA – downstream antisense primer or random hexamer and RT to make complete cDNA copy of RNA molecule †¢ Use cDNA, DNA polymerase, and a downstream primer in conventional PCR – extension leads to double-stranded DNA †¢ Regular PCR of dsDNA Wednesday, July 24, 2013 Applications of RT-PCR †¢ Detection ofShow MoreRelatedFiber Sensors For Bio Sensing1289 Words   |  6 Pagesdetection of the analyte. This analyte has a function to join physicochemical detectors and the components that are biological. A sensitive biological element includes cell receptors, organelles, enzymes, tissues, antibodies, microorganisms and nucleic acids. This element that is sensitive biological is a kind of bio-mimetic or a biological material which binds with the analyte during the study. There is another way to create biological sensitive elements and that is biological engineering throughRead MoreScience Study Of The Natural World1270 Words   |  6 Pagesneutrons Adhesion - the attraction that sticks two different materials together Surface Tension - the force in liquids that attempts to minimize surface area Electrolysis - the process where an electric current is passed through an ionized solution Amino Acid - the building blocks of proteins Polymerization - the process that creates polymers Catalyst - a material that speeds up a chemical reaction Activation Energy - the minimum amount of energy needed to start a chemical reaction Denature - remove naturalRead MoreWhat Is The Purity Check Of DNA?982 Words   |  4 Pagesbut are generally higher, which may mean that there are traces of RNA present in the sample since the expected value for RNA is around 2.0. 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Given that the nucleic acid aptamers function in vivo across blood plasma, it limitations has proven not to be efficient and accurate in cancer diagnosis. Due to its polynucleotides nature, n ucleic acid aptamers are naturally susceptible to enzymes degradation by exo-nucleases or enod-nucleases, leading to a reduced in vivo circulatory half-life [16]. The use of aptamers in animal models of disease and in human is seen to have reduced stability of natural nucleic acids in biological media [17]Read MorePollution from Synthetic Dyes1379 Words   |  6 Pagesforbidden with normal atmospheric triplet oxygen. 1.3.4 Sulfide Analysis The formation of methylene blue after the reaction of hydrogen sulphide with dimethyl-p-phenylenediamine and iron (III) at pH 0.4 – 0.7 is utilized to focus by photometric measurements. Sulfide concentration in the range of (20 ppb to 1.5 ppm). 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The identification based on enzyme ÃŽ ²-glucuronidase (product of gene uidA) specific to 94-97% of E.coli is monitoredRead MoreThe Effect Of Water Temperature On The Health Of An Estuary1661 Words   |  7 Pagesmay occur naturally, for example in shallow water overlying seagrass areas, higher than expected pH values may indicate blooms of phytoplankton or macroalgae. What factors affect PH level? Pollution generated from rain events, such as acid runoff from exposed acid sulphate soils on land, can drain into an estuary and reduce pH values dramatically. Alternatively, biological processes of photosynthesis associated with plants and algae can raise pH levels well above the normal marine range through removal