Nucleic acids blotting

The surface of each layer has two types of single stranded arms (e.g. one 3 — 5 and one 5 — 3 strand) which can bind to other monomers to render 1st-and 2nd-generation dendrimers 25 and 26. Therefore the molecular scaffold grows exponentially with each sequential layer of hybridization. If an oligonucleotide contains a sequence complementary to those at the surface of these networks it should be hybridized. The remaining free sequences from the other type of arms then bind in a standard nucleic acid blot (after they are bound to... 

Explain the purposes of protein and nucleic acid blotting. ... 

Both Parts I and II have been completely rewritten and reflect the many advances in biochemistry-molecular biology theory and techniques. Especially noteworthy have been the technical advances in chromatography (perfusion, FPLC, bioaffinity), electrophoresis (pulsed gel, capillary, nucleic acid sequencing), spectrophotometry (nmr, ms, and diode array detectors), and molecular biology (microsequencing of proteins and nucleic acids, blotting, restriction enzymes). 

Another common enzyme used m these procedures is alkaline phosphatase. Alkaline phosphatase will cleave phosphates off of a donor molecule, which then in turn acts as a mediator of a color change involving a third molecule. This system is often used because alkaline phosphatase can create more of the color-producing molecules per enzyme molecule than can peroxidase, resulting in better sensitivity. Alkaline phosphatase systems are especially sensitive for examining protein or nucleic acid blots with enzyme labels. The problem when examining tissue is the presence of the endogenous enzyme m the tissues examined. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

Oser, A., Roth, W.K., and Valet, G. (1988) Sensitive non-radioactive dot-blot hybridization using DNA probes labeled with chelate group substituted psoralen and quantitative detection by europium ion fluorescence. Nucleic Acids Res. 16, 1181-1196. 

Both the ELISA and Western blot suffer from the problem that antibodies may not appear in an aposed individual s blood until months after the initial exposure. Methods for using PCR to screen blood samples for HIV are being developed, PCR amplification of the HIV provirai DNA 7-ovides the ability to detect HIV at earlier stages of infection, because the viral nucleic acid is r resent immediately upon exposure. It is used to detect HIV infection in newborns whose mothers are HIV positive. 

---