Recent Changes

Monday, November 3

  1. page Co-Amplification at Lower Denaturation Temperature – Polymerase Chain Reaction (COLD-PCR) edited ... Co-Amplification at Lower Denaturation Temperature - Polymerase Chain Reaction Basic Descript…
    ...
    Co-Amplification at Lower Denaturation Temperature - Polymerase Chain Reaction
    Basic Description
    ...
    Brian Senajor
    Co-amplification

    Co-Amplification at Lower Denaturation Temperature – Polymerase Chain Reaction (COLD-PCR)
    Basic Description
    Co-amplification
    at lower
    ...
    a modified polymerase chain reaction or PCR.polymerase. The goal
    ...
    the denaturation stagestage, which separates
    ...
    strands. Next anthe intermediate annealing
    ...
    and the wildtypewild type of an
    ...
    forming heteroduplexes. Next comesNext, the melting
    ...
    DNA present. LastlyThe last is the extension stage. The extension stage
    ...
    performed with DNA polymerase. DNA polymerase will extendextends the primer into a complimentaryacomplimentary strand of
    Purpose
    The purpose of this technique is to amplify a low level mutation that is not present in a large amount of a genome’s alleles. A COLD-PCR is able to selectively amplify these mutated strands and separate them out of the rest of the alleles so that they can be studied. This is important because a normal PCR will not selectively amplify, and instead amplifies everything equally. This would make it very difficult to study a mutated sequence.
    Origins
    ...
    Li et al.,inal.in a Nature
    Current Research
    Zhuang Zuo et al., authors of “Application of COLD-PCR for improved detection of KRAS mutations in clinical samples” used COLD-PCR in order to detect KRAS mutations. KRAS is a member of the RAS oncogene family which is important in cell signaling. Mutations in the KRAS gene have been found to lead to cancer. This study used both a COLD-PCR as well as a standard PCR. It was found that the COLD-PCR was able to amplify the mutant strands at an amount 4-fold greater than a standard PCR. This is important to the study because the authors wanted to be able to focus on strands with a mutated KRAS gene and it is much easier to be able to isolate them during replication instead of amplifying everything [2].
    (view changes)
    8:50 am

Sunday, November 2

  1. page Li Fraumeni Syndrome edited Li Fraumeni Syndrome This syndrome is characterized by a mutations in the TP53 or CHEK2 tumor s…

    Li Fraumeni Syndrome
    This syndrome is characterized by a mutations in the TP53 or CHEK2 tumor suppressor genes, and as a result, individuals with this syndrome have much higher rates of cancer, specifically; sarcoma, breast, leukaemia, and adrenal gland cancers [1][2]. When damage to DNA is detected by the cell, a signal is sent to the CHEK2 gene which activates the TP53 gene; the TP53 gene transcribes a protein that initiates DNA repair and/or apoptosis. Mutations to this gene result in a missing regulatory pathway that allows cells with irregular DNA to progress through cell cycles normally and divide; this predisposes individuals with this syndrome to roughly 25 fold increased risk of developing a malignant tumor by the age of fifty[1]. Li Fraumeni syndrome is inherited through an autosomal dominant pathway, which means that an abnormal TP53 gene must be inherited from only one parent in order for the offspring to develop with this syndrome. The autosomal dominant pathway in this syndrome is characterized by a dominant negative mutation of the genes, which produce abnormal proteins that inactivate normal tumor suppressor proteins, so a heterogeneous individual will always have this syndrome.
    There are three characteristic variants of Li Fraumeni Syndrome; LFS1, LFS2, and a third locus on chromosome 1 related to an unidentified gene [2]. In the type I variant of Li Fraumeni, a mutation occurs on the TP53 gene directly [1][3]. This is usually associated with a premature stop codon which truncates the protein. There are eleven exons in this gene, and in most cases the mutations occur on exons five through eight, generally the remaining cases have mutations in either exon four or nine. The type II variant of Li Fraumeni contains one or more mutations on the CHEK2 gene, which regulates the TP53 gene[1][3]. Normally the CHEK2 gene receives a signal from ATM protein that detects DNA damage, and then activates the TP53 gene. A mutated CHEK2 gene is not able to convey the message that there is DNA damage to TP53 and the cell progresses through normal cell cycle with repair to DNA. Li Fraumeni type III is not characterized by a known gene, but the locus has been mapped to chromosome one [1].
    Many cancers, particularly breast cancer, are at a much higher risk in individuals with Li Fraumeni that the normal population with ~100% lifetime risk in females and 73% in males to develop cancer.
    {Li Fraumeni Flowchart.jpg}
    Fact Sheet:
    Name of Disease: Li-Fraumeni Syndrome
    Root cause of Disease: Irregularities in the normal function of the TP53 gene, which is responsible for initiating DNA repair mechanisms and/or apoptosis when DNA damage is detected.
    Affected Cell Types: Cell types involved in rapid division as well as nerve tissues are at a higher risk. The result is an amplified risk of all types of cancer, with higher rates among already high risk areas (breast, blood, sarcoma, adrenal gland, and brain cancers).
    Historical Background: The syndrome was first characterized by Frederick Pei Li and Joseph F. Fraumeni Jr., of whom the disease is named after.
    Common Symptoms: Several types of cancer, cancer development at a young age, cancer that appears several times throughout the lifetime of the affected individual.
    Standard Treatments: Genetic counseling and genetic testing are used to confirm the syndrome. Once a positive diagnosis is determined, regular cancer screenings are preformed. Radiation therapy is not done in an effort to reduce secondary radiation induced malignancies. Prophylactic mastectomy is an option to reduce the risk of breast cancer.
    Current Research: Current research is focused on early detection of cancer in LFS patients, and non-radiation treatment of these cancers. Physicians at Dana-Farber have been able to detect three types of cancer in asymptomatic LFS patients using PET-CT and full body MRI scanning techniques. There is also evidence that the types of breast cancer that arise in LFS are generally the types that can be treated by a targeted drug (monoclonal antibody that decreases the expression of HER2 gene), Trastuzumab (Herceptin) [4].
    References
    [1] Varley, J. (2003). Germline TP53 Mutations and Li-Fraumeni Syndrome. Hum. Mutat. 21 (3). 313-320
    [2] Cotran, R. Kumar, V. Collins, T. (2010). Robbins Pathologic Basis of Disease. (8) pp 288-290
    [3] Birch, JM. Harley, AL. Tricker, K. Prosser, J. Condie, A. Kelsey, A. Harries, M. Jones, P. Binchy, A. Crowther, D. Craft, A. Eden, O. Evans, D. Thompson, E. Mann, J. Martin, J. Mitchell, E. Santibanez-Koref, M. (1994). Prevalence and Diversity of Constitutional Mutations in the P53 Gene Among 21 Li-Fraumeni Families. Cancer Res. 54. pp 1298-304
    [4] Hudis, CA. (2007). Trastzumab—Mechanism of Action and Use in Clinical Practice. N Engl. J Med. 357 (1). 39-51

    (view changes)

Saturday, November 1

  1. page Confocal laser scanning microscopy (CLSM) edited Confocal Laser Scanning Microscopy (CLSM) Basic Description and Purpose ... selected focal pla…
    Confocal Laser Scanning Microscopy (CLSM)
    Basic Description and Purpose
    ...
    selected focal plane.plane [4]. The process
    ...
    of the specimen.specimen [4]. This process
    Operation
    ...
    of the sample.sample [4].
    {Confocal_laser_scanning_microscopy.jpg}
    http://en.wikipedia.org/wiki/File:Confocalprinciple_in_English.svg
    ...
    The original idea of this type of microscopy was patented by Marvin Minsky in 1957, but not until three decades later was it actually applied as the development of lasers was still in process. This type of microscopy is very beneficial in optical diseases, as it can break down the eye layer by layer to find infected areas. It also can be used for determining the age of Magdalen papyrus, which is a type of Egyptian paper.
    Recent Research
    The first recent articleIn a study investigating the sterilization of Enterococcus faecalis infected dentinal tubules, CLSM was using Three dimensional microvascular measurements in human endometrium using optical slices from laserscanningconfocal microscopy (LSCM) by Manconi, Frank. This was an article based on determiningused as a noninvasive technique to determine the structureefficacy of sterilization [1]. Infection of dentinal tubules was accomplished by centrifugation of the microvascular environment inbacterial suspension into the human endometrium.tubules. The endometrium has not been study through this type of microscopy and they wantinoculation procedure was followed by exposure to get a better understandingseveral concentrations of the functional importance of the tissue.
    The next article
    NaOCl sterilization solutions. Viability staining coupled to CLSM was determiningused to determine the developmentproportion of living a dead bacteria in the reproductive system of Echinostoma paraenseitubules layer by layer [1].
    Uranium exhibits fluorescent properties,
    in Mesocricetus auratus analyzed by lightbiological samples, cells, and confocalscanninglaser microscopy written by Souza, Joyce. This articlebiofilms, it is about the maturation of significant interest to determine the reproductive systemrole of Echinostoma paraensei worms grownuranium in an early infection of Mesocricetus auratus. Recovered flukes stained with hydrochloric carmine were preserved as whole mounts and then analyzed by confocal scanning laser microscopy.
    The final article used Intravitreal Concentrations of a Near-Infrared Fluorescence-Labeled Biotherapeutic Determined In Situ Using Confocal Scanning
    biological systems [2]. Laser Ophthalmoscopy, written by Anthony Basile. The purposeInduced Fluorescence Spectroscopy (LIFS) provides evidence of this researchuranium fluorescence emission, but no spatial localization can be determined. CLSM was used coupled to LIFS in order to determine spatially within biological systems where uranium was occurring. This research provided evidence of extracellular and intracellular compartmentalization of uranium in biological systems.
    Microfiltration of solutions through membranes result in decreased performance of membrane filtration over time due to membrane fouling [3]. Membrane fouling,
    the pharmacokineticsdeposition of ophthalmic biotherapeuticssolute particles onto the membrane surface regardless of nominal pore size, is a major obstacle in human vitrous humor, becausethe way of high transparency from NIR (Near infrared light), soproliferation of this technology. CLSM coupled to fluorescence staining was used to develop an entire cross-sectional profile of microfiltration membranes as fouling occurred. a pore blockage model is currently being developed based on this research [3].
    References
    [1] Ma, J. Wang, Z. Shen, Y. Haapasalo, M. (2011). A New Noninvasive Model to Study the Effectiveness of Dentin Disinfection by Using Confocal Laser Scanning Microscopy. Journal of Endodontics. 37(10). pp 1380-1385
    [2] Brockmann, S. Dresden. Grossmann, K. Arnold, T. (2014). Spatially Resolved Analyses of Uranium Species Using a Coupled System Made Up of Confocal Laser-Scanning Microscopy (CLSM) and Laser Induced Fluorescence Spectroscopy (LIFS). Internationale Zeitschrift fuer Kernenergie. 60(1). pp 31-34
    [3] Marroquin, M. Vu, A. Bruce, T. Powell, R. Wickramasinghe, S. Husson, S. (2014). Location and Quantification of Biological Foulants
    in placea Wet Membrane Structure by Cross-Sectional Confocal Laser Scanning Microscopy. Journal of it.Membrane Science. 453. pp 282-291
    [4] Paddock, S. (1999). Confocal Laser Scanning Microscopy. Biotechniques. 27 pp 992-1004

    (view changes)
  2. page Co-Amplification at Lower Denaturation Temperature – Polymerase Chain Reaction (COLD-PCR) edited ... Co-Amplification at Lower Denaturation Temperature - Polymerase Chain Reaction Basic Descript…
    ...
    Co-Amplification at Lower Denaturation Temperature - Polymerase Chain Reaction
    Basic Description
    Edited by, Brian Senajor
    Co-amplification at lower denaturation temperature – polymerase chain reaction or COLD-PCR is a modified polymerase chain reaction or PCR. The goal of COLD-PCR is to amplify certain alleles from a mixture of alleles. This differs from a regular PCR because a regular PCR amplifies all alleles present with no preference given to certain ones. This allows for easier detection of low level mutations in a genome. The principle behind COLD-PCR is that single nucleotide mismatches slightly alter the melting temperature of double-stranded DNA. This technique is similar to that of PCR except for the temperatures used. The first stage is the denaturation stage which separates the double stands into single strands. Next an intermediate annealing stage is used to allow the hybridization between the mutant and the wildtype of an allele to anneal to one another forming heteroduplexes. Next comes the melting stage and since the heteroduplexes melt at lower temperatures they are denatured at the critical temperature instead of the melting temperature. This causes the mutant strands to be replicated while the normal strands are not replicated because they do not melt. The next stage is the primer annealing stage where primers will anneal to the single strands of DNA present. Lastly the extension stage is performed with DNA polymerase will extend the primer into a complimentary strand of DNA to the template [1].
    Purpose
    (view changes)
    12:55 pm

Friday, October 31

  1. page Coomassie Blue Staining edited ... Current Research Coomassie Blue Staining technique is used in many biology research studies. …
    ...
    Current Research
    Coomassie Blue Staining technique is used in many biology research studies. In a research experiment, coomassie blue staining of proteins were compared to SYPRO Ruby (SR) staining with BioSafe and Neuhoff (NCCB) in one and two dimensional electrophoresis. In addition, the sensitivity of protein proteomes were measured. The second dimensional gel electrophoresis was used to analyze mouse and arabidopsis thaliana proteomes (Gauci et al, 2013). The experimental data yield that the Coomassie blue staining showed a lower abundance of protein in comparison to SR. Both coomassie blue staining and SR did not depict a significant level of sensitivity after the proteins were optimized overnight (Gauci et al, 2013). The mouse and arabidopsis thaliana proteomes were detect using the NCCB (Gauci et al, 2013). When Coomassie Blue was used with NCCB the protocol resulted in a more sensitive protein detection. The experiment research determined that the Coomassie Blue stain can depict sensitivity of the proteins that were optimized overnight (Gauci et al, 2013).
    ...
    column. In additionaddition, the researcher
    ...
    al, 2012).
    In another research study, coomassie blue dye was used to detect polyomavirus virion found in infected cells of mouse such as VP1 protein and cellular complexes (Hornikova et al, 2011). The mouse polyomavirus cells were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The results determined that bands were not present in the blue coomassie gel. The researchers stated that the BN-PAGE was beneficial tool to use to analyze different types of complexes formed by proteins. In addition, the method of using blue coomassie dye to detect new cellular proteins that are involved in virion assembly was not suitable (Hornikova et al, 2011). The analysis determined that the protein samples were made from viral proteins. The BN-PAGE was beneficial to detect the VP1 protein found in the nuclear lysates of the infected cells. The BN-PAGE detected several forms of VP1 proteins in the infected mouse cells (Hornikova et al, 2011). The coomassie blue stain dye was not proven to effective in this experiment. The coomassie blue stain dye was effectively used in Gauci et al experiment and Mekaoui et al experiment. The coomassie blue stain dye can be used to measure the proteins and protein sensitivity.

    References:
    ...
    Info Carol Roth . Roth.
    from http://www.carlroth.com/website/de-ch/pdf/Coomassie_E.pdf
    Coomassie Brilliant Blue R-250 and G-250 Dyes (2014). In Thermo Scientific Pierce Protein Biology .
    from http://www.piercenet.com/product/coomassie-brilliant-blue-r-250-g-250
    StainingGauci, V. J., Padula, M. P., & Coorssen, J. R. (2013, September). Coomassie blue staining
    for high sensitivity gel-based proteomics. Journal of Proteomics, 90(2), 96-106.
    Horníková, L., Man, P., & Forstová, J. (2011, December). Blue native protein electrophoresis
    for studies of mouse polyomavirus morphogenesis and interactions between the major capsid protein VP1 and cellular proteins. Journal of Virological Methods, 178(1-2), 229-234 .
    Mekaoui, N., Chamieh, J., Dugas, V., Demesmay, C., & Berthod, A. (2012, April 6).
    Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography. Journal of Chromatography A, 1232, 134-141.
    Staining
    Protein Gels
    ...
    In National Diagonistics Elecctrophoresis.DiagonisticsElecctrophoresis.
    from https://www.nationaldiagnostics.com/electrophoresis/article/staining-protein-gels-coomassie-blue
    (view changes)
    12:40 am

Thursday, October 30

  1. page Coomassie Blue Staining edited ... Coomassie Blue dyes were first produced in 1913. The dyes are similar to a wool dye used by Le…
    ...
    Coomassie Blue dyes were first produced in 1913. The dyes are similar to a wool dye used by Levinstein Ltd. The man who produced these dyes is, Max Weiler. The name Coomassie comes from a region in Ghana by the same name (Kumasi). Today there are more than 40 Coomassie dyes and all are trademarked at Imperial Chemical Industries. These dyes are very important to biochemical research and are still used today by many scientists.
    Current Research
    Coomassie Blue Staining technique is used in many biology research studies. In a research experiment, coomassie blue staining of proteins were compared to SYPRO Ruby (SR) staining with BioSafe and Neuhoff (NCCB) in one and two dimensional electrophoresis. In addition, the sensitivity of protein proteomes were measured. The second dimensional gel electrophoresis was used to analyze mouse and arabidopsis thaliana proteomes (Gauci et al, 2013). The experimental data yield that the Coomassie blue staining showed a lower abundance of protein in comparison to SR. Both coomassie blue staining and SR did not depict a significant level of sensitivity after the proteins were optimized overnight (Gauci et al, 2013). The mouse and arabidopsis thaliana proteomes were detect using the NCCB (Gauci et al, 2013). When Coomassie Blue was used with NCCB the protocol resulted in a more sensitive protein detection. The experiment research determined that the Coomassie Blue stain can depict sensitivity of the proteins that were optimized overnight (Gauci et al, 2013).
    In another experiment, coomassie blue G-250 was purified using a counter current chromatography multiple dual mode which allowed impurities to be removed (Mekaoui et al, 2012). An image of coomassie blue G-250 is shown in Figure 2. The research experiment measured 200 mg of purified coomassie blue extracted from 1 g of crude coomassie blue using 150 mL of butanol, 70 mL of heptane, and 200 mL of water (Mekaoui et al, 2012). The results of the experiment indicated that the counter current chromatography could not purify the coomassie blue dye because the coomassie blue dye sample was continuously trapped in the counter current chromatography column. In addition the researcher suggested that the solvent used in the experiment increased the levels of separation in the coomassie blue G-250 sample (Mekaoui et al, 2012).

    References:
    Coomassie Brilliant Blue and Mechanisms of Protein Staining and Bradford-Assay (n.d.). In Technical Info Carol Roth .
    ...
    Coomassie Brilliant Blue R-250 and G-250 Dyes (2014). In Thermo Scientific Pierce Protein Biology .
    from http://www.piercenet.com/product/coomassie-brilliant-blue-r-250-g-250
    ...
    Diagonistics Elecctrophoresis.
    from https://www.nationaldiagnostics.com/electrophoresis/article/staining-protein-gels-coomassie-blue

    (view changes)
    11:54 pm
  2. page Coomassie Blue Staining edited ... Protocol of Making a Protein Stain (Coomassie R-250 Brilliant Blue) 1. Obtain 45% Methanol (r…
    ...
    Protocol of Making a Protein Stain (Coomassie R-250 Brilliant Blue)
    1. Obtain 45% Methanol (reagent grade), 10% Glacial Acetic Acid, 45% water, and 3g/L Coomassie R-250 Brillant Blue
    ...
    45% water
    3.

    3.
    Dissolve about
    ...
    450mL methanol.
    4.

    4.
    Filter the
    ...
    the solution
    References

    References

    Coomassie Brilliant Blue R-250 and G-250 Dyes (2014). In Thermo Scientific Pierce Protein Biology .
    from http://www.piercenet.com/product/coomassie-brilliant-blue-r-250-g-250
    (view changes)
    6:49 pm

More