Genomic DNA isolation

Posted on Thu, Nov 24, 2016

In bacteria there are two main types of DNA—genomic and plasmid. Plasmid DNA is unique to bacteria. Eukaryotic cells don't typically have plasmid DNA unless it was put there by transfection for experimental purposes. The most important goal when isolating nucleic acids is to obtain the highest purity genetic material possible. When isolating genomic DNA it is important to remove plasmid DNA and RNA from the sample. Similarly, sometimes an experiment calls for the isolation of plasmid DNA, and the selective removal of genomic DNA is necessary. Also, some commercial RNA isolation kits include gDNA eliminator spin columns to remove genomic DNA from the isolate. 

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Choosing the appropriate DNA extraction protocol

Posted on Thu, Nov 17, 2016

 There are three general steps to DNA extraction

  1. celllysis and deactivation of DNAases
  2. Removal of contaminating molecules: proteins, polysaccharides, salts, other nucleic acids
  3. Recovery of DNA
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In-vitro diagnostic IVD coatings for magnetic particles

Posted on Thu, Nov 10, 2016

In-vitro diagnostic assays are used to diagnose infection or disease by specifically targeting a unique surface antigen or DNA sequence. Traditionally it was necessary to increase microbe density by culturing the sample for a few days under strict laboratory conditions. This was an important step toward obtaining enough genetic material for accurate analysis by qPCR.

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Chemiluminescence vs Fluorescence

Posted on Thu, Nov 03, 2016

  

Chemiluminescence and fluorescence seem like they are the same thing, especially when using them as tracking strategies for magnetic separation in biosensors or in-vitro diagnostic assays. But, they are not the same. Yes, they both give off a photon as an electron relaxes from a higher energy state to a lower energy state, but the difference lies in the method used to excite that electron to a higher energy state in the first place.  In fluorescence the electron is kicked up to a higher energy state by the addition of a photon. In chemiluminescence the electron is in a high-energy state due to the creation of anunstable intermediate in a chemical reaction. Light is released when the intermediate breaks down into the final products of the reaction.

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A magnetic molecularly imprinted polymeralternative to streptavidin magnetic beads

Posted on Thu, Oct 27, 2016

 

Streptavidin magnetic beads are used to isolate biotinylated biomolecules

The affinity between streptavidin and biotin is one of the strongest non-covalent interactions known in nature. It is extremely specific and is unperturbed by extreme changes in temperature, pH, and detergent. It is therefore a very useful tool in biotechnology. For example, the system is commonly used in magnetic separation. Target molecules are biotinylated, which means that a biotin molecule is covalently attached to the protein, nucleic acid, or other molecule of interest. Streptavidin-bound magnetic particles are incubated with a solution containing the biotinylated target molecule for a sufficient time for the streptavidin-biotin affinity to form. The complexes can then be isolated from solution by magnetic separation.

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A chemiluminescence assay for the detection of the Zika virus

Posted on Thu, Oct 20, 2016

The Zika virus (ZIKV) is emerging as a global public heath threat. It has been linked to the development of microcephaly in newborn babies whose mothers’ were infected by ZIKV during pregnancy. Recently, it has been shown that the virus can be sexually transmitted.  The clinical signs in adults include fever, rash, joint pain, and conjunctivitis, which are common clinical signs for a general viral infection. This makes clinical diagnosis difficult without a specific immunoassay. The current methods for ZIKV detection are qPCR (quantitative polymerase chain reactions) or ELISA (enzyme-linked immunosorbent assay). Each of these is time consuming and the reagents can be expensive. As more people become infected and the virus spreads throughout the population it will become necessary to find a cheaper and faster alternative assay for detection of ZIKV. A magneto-actuated chemiluminescence immunoassay for the Zika virus has been developed. The demonstrated limit of detection is comparable to qPCR.

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Different types of Immunoassays

Posted on Thu, Oct 13, 2016

An immunoassay capitalizes on the specificity of the antibody-antigen binding found naturally in the immune system. The assay can be used to identify the presence of pathogens in a clinical sample, or it can be used to measure the amount of a target biomolecule. If the goal of the immunoassay is to isolate a specific molecule then a separation system is needed. When the isolation is achieved by magnetic separation using a magnetic particle it is called a magneto-actuated immunoassay. The most common particle used in these assays is made of a core of magnetite that is coated with a biologically compatible material, and chemically modified by the attachment of antibodies. However, before designing a magnetic particle for an immunoassay one must decide which type of immunoassay best fits the goals of the experiment.

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A magneto-actuated ELISA to quantify CD4+ lymphocytes in whole blood

Posted on Thu, Oct 06, 2016

CD4+ lymphocytes are of particular interest in patients infected with Human Immunodeficiency Virus (HIV). A devastating effect of this retroviral infection is a progressive loss of CD4+ cells. These cells are crucial to a healthy immune system. When the level of CD4+ cells fall below 200cells/uL whole blood the patient is diagnosed with Aquired Immune Deficiency Syndrome (AIDS).  Therefore, the number of CD4+ cells in these patients is a good way to determine when to start antiretroviral treatment, and to administer drugs to protect against opportunistic pathogens.  Currently, the most accepted way to measure the CD4+ concentration in whole blood is to use flow cytometry. However, this technology is extremely expensive and requires trained technicians to operate and maintain the machine. An alternative method is needed in resource poor areas. This alternative method would ideally be implemented at the point of care to avoid transportation of samples to and from a laboratory. One such recently developed technology is a magneto-ELISA to detect the concentration of CD4+ lymphocytes in whole blood.  The detection system relies on magnetic separation to obtain an isolated population of CD4+ cells. It has a demonstrated limit of detection of 50 Cd4+ cells per μL, which is sensitive enough to diagnosis AIDS.

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DNA capture using magnetic nanoparticles

Posted on Thu, Sep 22, 2016

 

Efficient methods for DNA detectionin clinical, environmental, and experimental samples are constantly in demand. In a clinical sample, DNA capture and identification can be essential to the diagnosis of disease. In public heath and environmental situations it can be used to identify contamination of food or water. DNA collection and characterization is constantly expanding our ability to answer experimental questions. DNA capture is a mainstay of modern biotechnology. Traditional techniques rely on affinity columns, centrifugation, and multiple washing steps. Newer methods are based on magnetic separation. In the beginning,magnetic separation was limitedto packing a column with magnetic material and running a solution through it. As nanotechnology evolved it became possible to use mobile solid support systems such as magnetic nanoparticles.

 

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IVD assays for point of care malaria diagnosis

Posted on Thu, Sep 15, 2016

 

An in vitro diagnostic product (IVD) is any reagent, device, system, or part of a system used outside of the body to diagnose a disease or infection. The IVD can be used to detect DNA/RNA, microorganisms, or protein. This can be in a laboratory setting or in a “point of care setting.” Point of care (POC) is beneficial because it removes the need to send a sample to a laboratory for testing. Therefore, the time between sample collection and diagnosis is significantly reduced. Point of care IVD is especially useful in resource-poor settings where laboratories are located far away and there is a lack of good communication or transportation infrastructure.      

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