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One-step synthesis of fluorescent carbon dots for …
One thing I should confess is that this example has one bit of fiction - you can'treally obtain usable sequence information that close to the end of the primer, unlessyour ddNTP/dNTP ratios are quite high. If you increased that ratio, you would makeit more difficult to read sequence 200 to 300 nucleotides further down, because mostof the synthetic products would have terminated before that point.
It is possible to obtain labeled products in one of two ways:
In this case, one of the dNTP substrates is radiolabeled (or chemically labeled)so that the synthetic products are marked internally, and possibly in many placesat once. The multiple sites of incorporation mean that the product will have a higherspecific activity.
An advantage of this is that only reactions involving the annealing of the oligonucleotidewill be labeled. A disadvantage is that at most one labeled atom is incorporatedinto the synthetic product, so the specific activity is low.
The technology of DNA labeling has changed in the last dozen years, so thatthere are many more options.
P-32 phosphate labeling has many advantages and several disadvantages. Thehalf-life of P-32 is approximately 14 days, and so it is possible to label DNA toa very high specific activity (meaning that a small number of moles of product generatea large number of radioactive disintegrations per minute). One disadvantage of P-32is that it produces a penetrating burst of beta particles (electrons) that generatea "wider" signal on a piece of film. In the schematic diagram below, youcan see how this effect works:
Synthesis and Characterization of Tb 3+-Doped Gd 2 …
Then split the sample into four aliquots, in tubes labeled "G", "A","T" and "C" and add the following substrates to the respectivetubes:
"G" tube: All four dNTPs, including one that is labeled, plus
"A" tube: All four dNTPs, including one that is labeled, plus
"T" tube: All four dNTPs, including one that is labeled, plus
"C" tube: All four dNTPs, including one that is labeled, plus
When a polymerase (e.g. Klenow fragment) is added to the tubes, the syntheticreaction proceeds until, by chance, a dideoxynucleotide is incorporated instead ofa deoxynucleotide. This is a "chain termination" event, because there isa 3' H instead of a 3' OH group. Since the synthesized DNA contains some radiolabeled(or chemically labeled) substrates, the products can be detected and distinguishedfrom the template.
If, for example, we were to look only at the "G" reaction, there wouldbe a mixture of the following products of synthesis:
makes amachine that we use in Sc4108. They have made a program called EditView, which , and which shows the types of chromatograms one obtains from the laserreadout.
. In this method, the oligonucleotide primers are end-labeled withfour different fluorescent dyes, and four separate synthetic reactions are conductedin the presence of dNTPs and ddNTPs:
Synthesis and Fluorescence Properties
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