Flow FISH is a method for determining the lengths of telomeres which is common nowadays due to its versatility, its more rapid and its able to process samples which contain few cells (Lansdorp,1996). The major limitation of flow-FISH is the requirement of the equipment to do the analysis which is expensive, the configuration of the equipment (flow cytometer) and the nature of the probe used for hybridization is also expensive and requires a well trained personnel. Flow-FISH involves hybridisation of telomeric DNA of fixed sample cells with a fluorescently labeled peptide-nucleotide probe with sequence which is complementary to the telomere repeat DNA sequences. PNA is a synthetic polymer containing nitrogen bases similar to those of DNA (Kapoor and Telford, 2004). The backbone of PNA consists of N-(2-aminoethyl)-glycine units linked by peptide bonds unlike DNA backbone. Nitrogen bases are linked to the backbone by methylene carbonyl bonds instead of DNA N-glycoside bonds. Melting temperature of the PNA-DNA hybrid is higher than of DNA-DNA hybrid (Egholm et al., 1993), allowing hybridisation at high temperatures. The probe is labelled with dyes which are excited in the ‘blue’ visible light range (440-490 nm) since flow cytometers have a blue laser. Most often, fluoresceine isothiocyanate (FITC) is used as a label. If flow cytometer is equipped with a red laser, fluorescent labels with excitation peaks in the red visible range are used to label the PNA probe. To control cell cycle phase, the DNA is stained with dyes capable of binding to the DNA in a quantitative fashion such as propidium iodide (PI) or 4,6-diamidino-phenylindole (DAPI) (Nielsen et at.,1991). DAPI is easier to use, just like PI, it will bind to RNA, but the emissi...
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...ge, inhibitors accumulate, or even inactivation of the taq polymerase has started which may affect the efficiency of the PCR reaction. Fluorescence readings at early amplification cycles will gauge the amplified template quantity where the reaction is much more reproducible from sample to sample than at the endpoint. Use of a standard curve is a direct and accurate approach for analyzing the quantity of DNA .Standard curve is usually prepared from a dilution series of template whose concentration is known. The standard curve approach is used when it is important to the experimental design and objective of the project to measure the exact level of template in the samples. A variety of sources can be used as standard template such as plasmid which has a cloned gene of interest, genomic DNA, copy DNA, synthetic oligos, in vitro transcripts and total RNA besides others.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
completed, the tubes are stored at 4°C until analysis of the tubes. To alylize the PCR results with
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
Miller, Kenneth R. and Joseph S. Levine. “Chapter 12: DNA and RNA.” Biology. Upper Saddle River: Pearson Education, Inc., 2002. Print.
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
Inside the nucleus of our cells, our genes are on double-stranded molecules of DNA called chromosomes. At the top and bottom of the chromosomes are fragments of DNA known as Telomeres which defend our genes, give us the ability for our cells to divide, and hold secrets to how we age and how we get cancer. Telomeres are like the ends of shoelaces (because they keep the chromosomes’ ends from fraying). But when a cell divides, the Telomere gets smaller and shorter. When they get too short, the cell can’t divide. The Telomeres then become “senescent” or inactive. This shortening is linked with aging, cancer, and death-risk. Telomeres should also be compared to a bomb fuse. Without Telomeres, the main part of the chromosome would get smaller whenever the cell divides. This can cause a malfunction or cancer. An enzyme named telomerase adds bases to the ends of Telomeres. In young cells, telomerase keeps Telomeres from wearing down too much. But as cells divide repeatedly, there is not enough telomerase, so the Telomeres grow shorter and the cells age. Telomerase remains active in sperm and eggs, which are passed from one generation to the next. If reproductive cells did not have telomerase to maintain the length of their Telomeres, any organism with such cells would soon go extinct.
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
Q1Aamp DNA mini kit, leading to the second period being the amplification of 16s rDNA consensus sequence by PCR, particularly using the primers RW01 and DG74. The unknown sample is then taken for gel electrophoresis to confirm and purify the amplified 16s rDNA fragment, done in the third period of the experiment. Once the running of the gel is completed, a cut of the 370bp PCR fragment is taken, and is put for purification of 16s rDNA fragment by the QIAquick gel elution kit, allowing the DNA to elute at the bottom of the microcentrifuge tube
DNA damage is a proven biomarker of radiation effects. Cytogenetic techniques commonly employed for detecting DNA strand breakages include chromosome aberrations, micronucleus and comet assay under field and laboratory circumstances. In recent times, flow cytometry (FCM) has been widely used to quantify DNA damage since it offers the analysis of a high number of nuclei in a few minutes providing statistically reliable results in a short period of time. Ideally, all cells within an organism contain the same amount of DNA. DNA damage results from the breakage and rearrangement of chromosomes and from interference with the normal segregation of chromosome during cell division. Double strand breaks are the most important DNA lesions caused by ionizing radiation and other damages from exposure to genotoxic agents resulting in cells with an abnormally high or low DNA content. This variability can be detected as an increased coefficient of variation (CV) of cells in G0/G1 phase as measured by flow cytometry.
The G-rich and C-rich DNAs individually form the parallel G-quadruplex and I-motif, respectively, in the molecular crowding condition, and the 1:1 mixture folds into the parallel G-quadruplex and I-motif but does not form a duplex. The ITC measurements indicated that the thermodynamic stability (ΔG°20) of the duplex formation between the G-rich and C-rich DNAs in the noncrowding condition was −10.2 kcal mol-1, while only a small heat change was observed in the ITC measurements in the molecular crowding condition. These ITC results also demonstrated that the molecular crowding condition prevents any duplex formation between G-rich and C-rich DNAs. These results indicate that a structural polymorphism of the telomere DNAs is induced by molecular crowding in vivo [25]
5 test tubes were used for this lab, each label 1-5. Then we added 1ml of assign solution for each test tube. Tube 1 had DNA, 2 had RNA, 3 had the nucleic acids from the liver extract supernatant, 4 had water, and 5 had the unknown. 2ml of Dische’s reagent was then added to each test tube. All of the test tubes are then place in boiling water. After 15 minutes in the boiling water, we recorded the color of each of the different test tubes in a table.
Tsou, J. A., Hagen, J. A., Carpenter, C. L., & Laird-Offringa, I. A. (2002, August 05). DNA
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...
Write an essay explaining the continuity of life and how it is based on heritable information in the form of DNA and its transmission from one generation to another.