Electrophoresis using nucleic acid probes

Here a is a material-related constant independent of the matrix M. In terms of Eq. 3.2, the Ogston model predicts v = l,S = 2, and y =0. 

Modem biochemistry provides flexible and semi flexible water-soluble polymers having extremely well-defined molecular weights, including double-stranded DNA (dsDNA), single-stranded DNA(ssDNA), RNA, and complexes of surfactants with 

We begin with Barron, et al, who reported mobilities of restriction fragments in solutions of hydroxyethylcellulose, hydroxypropylcellulose, and linear poly-acrylamide(5,6). Barron, et al. found that dilute polymer solutions are effective separatory media for DNA fragments. Dilute polymer solutions had previously not been expected to be effective separatory media, because the theoretical models being invoked in the electrophoresis literature referred only to gels and nondi-lute solutions. In these models, interpenetrating polymer coils were claimed to form evanescent separatory pores. In dilute solution, polymer coils do not interpenetrate, so the hypothesized pores should not be present, and therefore there was expected to be no separation. Barron, et al. concluded that they had evidence for a new mechanism for DNA separation at low matrix concentration, a mechanism distinct from the pore formation mechanisms presumed active at large c(6). 

Barron, et al. examined the same set of restriction fragments in solutions of seven matrix polymers hydroxypropylcellulose M of 100, 300, and 1000 kDa), linear polyacrylamide M of 700-1000 and 1140 kDa), and hydrox-yethylcellulose (Mw of 1.3 and 1.76 MDa)(6). These matrix polymers were not monodisperse polydispersities extended from 2.5 to 12.4. 

Heller made a systematic exploration of polyacrylamides, hydroxypropylcellu-loses, methylcelluloses, hydroxyethylcelluloses, polyethylene oxides, and dextrans to find an optimal (low viscosity, high resolving power) polymer to use as a support medium(14). Dextrans are effective support media for dsDNA (10-10 bp) separations, and have lower viscosities than other matrix polymers with similar 

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Northern blotting was not named for its inventor, but as a companion technique that uses RNA rather than DNA as the test nucleic acid. RNA is transferred from the gel after electrophoresis onto a solid support followed by hybridization with a specific labeled probe. Because RNA molecules have defined lengths and are much shorter than genomic DNA, it is not necessary to cleave RNA before electrophoresis. However, because of the secondary structure of RNA, it is necessary to perform electrophoresis under denaturing... 

Gel electrophoresis is a powerful and versatile method to resolve mixtures of different nucleic acid molecules and allows the fractionated molecules (i) to be viewed directly, (ii) to be recovered in pure form or (iii) to be characterized directly by hybridization. Hybridization of the probes to fractionated DNA ( Southern technique ) (Southern, 1975) or fractionated RNA ( Northern technique ) (Al-wine et al., 1979) can be achieved after the transfer of the resolved molecules to a membrane, but in some cases also directly in the gel using oligonucleotide probes ( unblot ) (Purrello and Balazs, 1983 Tsao et al., 1983). The steps in these protocols are summarized in Table 9.1. Simultaneous extraction of DNA and RNA (Section 3.4.3) (Chan et al., 1988) may be advantageous when the mass of tissue available is small. 

With gel electrophoresis as a quantitative tool for size determination, the problem is more serious unlike nucleic acids, mucins do not have a natural uniform charge length ratio, and furthermore, unlike for unglycosylated proteins, sodium dodecyl sulfate does not bind uniformly. The technique appears to have some use, however, as a probe for possible mucin-protein interaction. ... 

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