DNA migration

In the first report, PAMAM dendrimers with primary amine surface groups were used [27]. It was found that complex formation is dependent both upon the size (generation) of the dendrimers used and the charge ratio between the (cationic and anionic species, i.e. ammonium groups on PAMAM to phosphate groups on DNA). Retardation of DNA migration was not observed with... 

Figure 5.9 Comparison of DNA damage induced by [Ru( s-p-cymene)Cl2(pta)] incubated at different pH values shown by DNA migration in an agarose gel. Reproduced by Permission of the Royal Society of Chemistry...

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

Most theoretical treatments of the gel electrophoresis of DNA molecules are based upon de Gennes (1971) concept of reptation. The migration of the nucleic acid is considered to occur in a tube formed by the polymer matrix of the gel, through which the DNA migrates rather like a... 

FIGURE 9.30 Fluorescence images of (a) a XI)NA and (b) a T4 DNA migrating in a nanopiUar region at 7 V/cm [351]. Reprinted with permission from the American Chemical Society. 

Because a DNA molecule is a flexible polymer, the assumption in the Ogston model that DNA migrates as an undeformable particle loses validity with increasing size of DNA. The Ogston model predicts that the mobility of DNA with Rg larger than the pore size of the gel will approach 0. However, it actually was observed experimentally that DNA with its Rg larger than the gel pore size keeps migrating. 

Molten 1% LGT agarose should be maintained at 40°C to aid uniform gel preparation. The thickness of the gel should be consistent between different slides to ensure uniform DNA migration. The gel should not flood the entire slide and the frosted section should be avoided. Ideally all gels should have the parameters of the coverslip. 

The retrovirus enters the cell through the CD4 molecule on the cell surface. Once inside, the virus is uncoated with the help of the reverse transcriptase enzyme, enabling the virus single-stranded RNA to be converted into DNA. The viral DNA migrates to the nucleus of the cell, where it is spliced into the host DNA with the help of the integrase enzyme. Once combined, the HIV DNA is called the provirus and is duplicated each time the cell divides. The protease enzyme assists in the assembly of a new form of the viral particles. 

The glass plate supporting the filter papers and gel is adjusted so that the ends of the filter paper are suspended in buffer and act as wicks. The buffer moves upward through the gel and into the wad of paper towels that act to absorb the buffer. The DNA migrates from the gel to the nitrocellulose filter that binds the single-stranded DNA fragments. 

An increase in DNA migration parameters indicates that the test substance has induced DNA strand-breaks, while a decrease suggests DNA-DNA and DNA-protein crosslinks. 

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