What is an oligonucleotide ligation assay? How does it work?
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I also don't know what you're referring to. I've never heard of something called an oligonucleotide ligation assay. Usually I stop myself before jumping in to say that something is wrong just because I've never heard of it, because there is plenty of stuff I've never heard of. But let me tell you what those words mean to me. Oligo means few, so an oligonucleotide is a string of nucleotides, like a primer. It's generally used as either a primer or a probe. Ligation is sticking something together. In the same context as oligonucleotides, that would generally mean subcloning something.\n\nThere are a lot of ways to check sequence, but none of them really involve ligating oligonucleotides. I know two ways to check for this, one vastly preferable to the other. Get primers flanking the region, PCR amplify, and sequence. Really, you can stop reading there and just do that. The other assay is for genotyping that has to be done often, where it is too expensive to sequence everytime. Get primers flanking the region. There will be one forward primer, but two reverse primers. Each of the reverse primers will match one of the polymorphisms, but one of them will have a longer tail. Run the products on a gel and differentiate the two by size.\n\nIt's a pain to do and doesn't work well. Use the first method.\n\nNow I'm going to look up "OLA" and see if I can learn something new today.
137653
There are different ways to sequence cDNA, probably the most common right now is PCR based:  an unlabeled primer is put into a PCR reaction with the CDNA, fluoroescent molecules attached to dNTPs (a different color for each) are added along with non-labeled dNTPs.  Addition of the fluorescent dNTPs stops the reaction so that different length fragments with different dNTPs at the end are obtained.   By analyzing the different lengths of these fragments by electrophoresis and looking at the color of the dNTP terminator, the sequence can be obtained.
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Restriction enzymes- they cut the phage's DNA so that the instructions within the DNA don't make any sense anymore and can no longer harm the bacteria. \n\nScientists are now using these restriction enzymes in laboratories to duplicate it in biotechnology testing
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figure out the efgfect of the enzyme. Or you can test DNA structure. Inhibitary enzymes have a blacked actionsite whereas activation enzymes have activation sites open to substrates
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If you have a purified prep of chromatin, you can use the spec to measure the amount of protein at 254 nm.  The problem is that this overlaps with DNA absorbance (260/280nm).  You can use a protein assay such as bradford or BCA to measure protein concentration.  Usually western blots are used to measure relative protein concetrations of specific proteins ie histone, actin.
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What's the size of your oligonucleotide and its concentration?  There's no recommended concentration for primers since you must input as much primer as your sample requires.  Determine your sequnce size and concentration first, then correlated it using: http://dna.chmcc.org/dnaseq/html/primconcalc.html and work on that.\n\nGet cracking.
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- with PCR you amplify a DNA sequence (like ATGGTAGCATGATC etc etc etc)\n- P = polymerase (thats the name of the enzyme that does the amplification\n- C = chain (there are actually two chains: first the chain of nucleotides ATGGAGACCCTAetc and second the chain of polymerization reactions)\n- R = the enzymatic amplification reaction\n- you can only do a PCR with a DNA double strand, because you need two starter oligonucleotides that bind to the initial template that you want to amplify; they resemble a short DNA sequence of the complementary (opposite) strand; they work best when they have a length between 18 and 20 nucleotides. When you do a PCR, you will most likely know the sequence of these oligonucleotides and you will order them from a company who designs them for you (you just give them the sequence you desire and they make them fo you)\n- so when you do a PCR, you have to offer the polymerase enzyme some material so that it can work: i.e. the template DNA that you want amplified, the starter oligonucleotides and especially the single nucleotides A, T, G, C\n\n- for RFLP you will need an enzyme that cuts the DNA strand\n- restrictions enzymes cut specific sites\n- they have names like BamHI EcoRI etc etc etc\n- example: BamHI cuts at the site GGATCC and only there\n- so when you have a DNA sequence that is, say 2000 basepairs long, and you have only one GGATCC, the enzyme will cut only once. If the DNA sequence was linear (i.e. it has two ends like a sausage) then you will get 2 DNA fragments. If your DNA is circular, you will get one fragment\n- so think in larger dimensions: if you cut a tremendously long DNA sequnece, you will have more GGATCC sites, consequently you will get more fragments.\n- so if you know your sequence, you can just do your simple math calculations and predict the fragments and their lengths: from the fist GGATCC to the second its, for example 10,010 basepairs, from the second to the third xxx and so on\n\n- R = restriction (cutting the DNA strand into pieces)\n- F = fragment (the pieces of DNA that arise when cutting the large one)\n- L = length (the different lentgs of DNA pieces that arise during the cutting)\n- P = polymorphism (referring to the fragment pattern when comparing them to the pattern of different samples)\n- the actual RFLP comparisons are made in the gel, because sometimes you don't have the exact DNA sequence of every DNA you want to analyze\n- for example, you cut the genomic DNA of two or more related bacterial species. And lets say you will know the exact sequence of only one of them. For that one you can make predictions about fragment lengths based on your calculations. For the others you can't, because maybe they have mutations in their sequences.\n- so when you compare the fragments to each other and to the one you initially precalculated, you might find that the other related species reveal different fragments. That means the sequences af the repated species have alterations in the site where the restriction enzyme was supposed to cut.\n- So the interpretation will be that specific DNA sites have an altered sequence to the sequence that was given to you\n- you can also make predictions about total mutations in the DNA, if you know how long the total DNA was and if you know how many restrictions sites there are and how many were not cut with a specific enzyme
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As far as using for detection, its very vulnerable to showing false positives from non-specific binding.  In addition, because of the relatively low level of mRNA, it can only be used for fairly abundant messages.  It is somewhat difficult to separate non-specific from specific binding, but of course it can be done.  In addition, it only shows the presence of message-it does not tell about the level of protein translation.
961312
Well, that is the Holy Grail of biochemistry and we've been dreaming of that ever since Dr. Pauling had me researching metabolic diseases, Lesch-Nyhan syndrome in particular.  The problem has been 1) finding the precise genetic coding of the fouled up part of the DNA and 2) devising a mechanism to clip out that nucleic base and replace it with the correct one (or series of bases).  Precision is, of course, vital or you'll kill the experimental animal (or, ultimately, human patient).
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Detection of specific DNA fragments by gel-transfer hybridization (Southern blotting). (A) The mixture of double-stranded DNA fragments generated by restriction nuclease treatment of DNA is separated according to length by electrophoresis. (B) A sheet of either nitrocellulose paper or nylon paper is laid over the gel, and the separated DNA fragments are transferred to the sheet by blotting. The gel is supported on a layer of sponge in a bath of alkali solution, and the buffer is sucked through the gel and the nitrocellulose paper by paper towels stacked on top of the nitrocellulose. As the buffer is sucked through, it denatures the DNA and transfers the single-stranded fragments from the gel to the surface of the nitrocellulose sheet, where they adhere firmly. This transfer is necessary to keep the DNA firmly in place while the hybridization procedure (D) is carrried out. (C) The nitrocellulose sheet is carefully peeled off the gel. (D) The sheet containing the bound single-stranded DNA fragments is placed in a sealed plastic bag together with buffer containing a radioactively labeled DNA probe specific for the required DNA sequence. The sheet is exposed for a prolonged period to the probe under conditions favoring hybridization. (E) The sheet is removed from the bag and washed thoroughly, so that only probe molecules that have hybridized to the DNA on the paper remain attached. After autoradiography, the DNA that has hybridized to the labeled probe will show up as bands on the autoradiograph. An adaptation of this technique to detect specific sequences in RNA is called Northern blotting. In this case mRNA molecules are electrophoresed through the gel and the probe is usually a single-stranded DNA molecule.
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Restriction enzymes, also called restriction nucleases (EcoRI in this example) , surrounds the DNA molecule at the point it seeks(sequence GAATTC). It cuts one strand of the DNA double helix at one point and the second strand at a different, complementary point (between the G and the A base). The separated pieces have single stranded "sticky-ends," which allow the complementary pieces to combine. \n\nThe newly joined pieces are stabilized by DNA ligase.\n\nEcoRI, one of many restriction enzymes, is obtained from the bacteria Escherichia coli.
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In order to perform in vitro analyses, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates the various cellular components into fractions containing soluble proteins; membrane lipids and proteins; cellular organelles, and nucleic acids. Precipitation by a method known as salting out can concentrate the proteins from this lysate. Various types of chromatography are then used to isolate the protein or proteins of interest based on properties such as molecular weight, net charge and binding affinity. The level of purification can be monitored using gel electrophoresis if the desired protein's molecular weight is known, by spectroscopy if the protein has distinguishable spectroscopic features, or by enzyme assays if the protein has enzymatic activity.\n\nFor natural proteins, a series of purification steps may be necessary to obtain protein sufficiently pure for laboratory applications. To simplify this process, genetic engineering is often used to add chemical features to proteins that make them easier to purify without affecting their structure or activity. Here, a "tag" consisting of a specific amino acid sequence, often a series of histidine residues (a "His-tag"), is attached to one terminus of the protein. As a result, when the lysate is passed over a chromatography column containing nickel, the histidine residues ligate the nickel and attach to the column while the untagged components of the lysate pass unimpeded.\n\n\n[edit] Cellular localization\n \nProteins in different cellular compartments and structures tagged with green fluorescent protein.The study of proteins in vivo is often concerned with the synthesis and localization of the protein within the cell. Although many intracellular proteins are synthesized in the cytoplasm and membrane-bound or secreted proteins in the endoplasmic reticulum, the specifics of how proteins are targeted to specific organelles or cellular structures is often unclear. A useful technique for assessing cellular localization uses genetic engineering to express in a cell a fusion protein or chimera consisting of the natural protein of interest linked to a "reporter" such as green fluorescent protein (GFP). The fused protein's position within the cell can be cleanly and efficiently visualized using microscopy, as shown in the figure opposite.\n\nThrough another genetic engineering application known as site-directed mutagenesis, researchers can alter the protein sequence and hence its structure, cellular localization, and susceptibility to regulation, which can be followed in vivo by GFP tagging or in vitro by enzyme kinetics and binding studies.\n\n\n[edit] Proteomics and bioinformatics\nMain articles: Proteomics and Bioinformatics\nThe total complement of proteins present in a cell or cell type is known as its proteome, and the study of such large-scale data sets defines the field of proteomics, named by analogy to the related field of genomics. Key experimental techniques in proteomics include protein microarrays, which allow the detection of the relative levels of a large number of proteins present in a cell, and two-hybrid screening, which allows the systematic exploration of protein-protein interactions. The total complement of biologically possible such interactions is known as the interactome. A systematic attempt to determine the structures of proteins representing every possible fold is known as structural genomics.\n\nThe large amount of genomic and proteomic data available for a variety of organisms, including the human genome, allows researchers to efficiently identify homologous proteins in distantly related organisms by sequence alignment. Sequence profiling tools can perform more speci
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It binds to a specific sequence of the gene. If the gene is present, it will bind, if not, it will not bind. Usually the probe also has a fluorescence end, therefore you can use a light to show that it did bind or not.
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Certain ligases which cut DNA at known sequence points are used to excise the gene of interest from a particular strand of DNA. This excised (or cut-out) gene is then incorporated into the DNA of a bacterium.  The bacterium, in the process of replication, will add the gene into its own plasmid and produce the protein. Alternatively, I believe viral vectors are also used to introduce genes into a particular host "factory" cell that then produces the required protein. Not totally informed on that part, though.
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I think I might be qualified to answer this question.\n\nDNA is separated in an electrophoretic field based on the size of the molecules.  \n\nImagine wet spaghetti strands (all different sizes) passing through a thick block of Swiss cheese.   Agarose electrophoresis is based on the ability of the smaller molecules to pass through holes easier than the larger molecules.\nThe smaller the mass, the more nimble it is, and the quicker it can manage all those "Swiss cheese" holes that agarose gels naturally have.\n\nOnce the DNA has run through the gel for a sufficient amount of time, you can stain the gel (actually you can stain the gel as the DNA moves through the gel too).  \n\nThe stain (the classic one is ethidium bromide, but there are others) has a propensity to squeeze itself between nucleotides on both strands.  When it is exposed to a transilluminator light  with a specific wavelength, the ethidium bromide will fluoresce and can be photographed for a permanent record.  \n\nAnalysis is typically based on running controls (positive and negative controls) and a size ladder along with the "unknown" PCR amplicon.  Usually I can predict the size of my unknown amplicon before I run the reaction. \n\nI can then choose a size ladder that will at least give me a rough idea of the size of my amplicon I expect. The ladder also allows me to observe any other false amplicons created during the PCR.  Additionally, some like to estimate how much PCR amplicon was generated in the reaction by comparing the intensity of the size ladder with the intensity of the amplicon. \n\nI hope I was able to help in answering your question.  Hoping YOU spread the good Karma!
447397
Restriction enzymes are cutting enzymes found in bacteria. They cut within the DNA molecule and are called restriction endonucleases. They recognize a specific sequence 0f DNA nucleotides and cut the molecules there, producing sticky ends. A missing gene is then added. Another enzyme, DNA ligase, attaches back the DNA molecule. So, of course you end up with two DNA strands. The new DNA produced is known as recombinant DNA. This technology is used in human therapy. Some examples are: human insulin to treat diabetes type I and human factor VIII for the treatment of hemophilia.
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One is a simple binding assay, in which the ligand (usually radiolabeled) is added to a hemogenate of the tissue-a number of washes are done, in the presence of cold ligand, in order to retain only that ligand that has bound with high affinity.\nThe others are all protein-based.  Western blotting with specific antibodies, immunohistochemistry to determine the presence and cellular localization.  RT-PCR or Northern blotting will tell you if the mRNA is present, but not if the receptor protein itself it there.