The membrane containing the transferred DNA is then soaked in a solution containing the radiolabeled probe. After transfer, the DNA fragments are arrayed by size on the solid support.Īt this point, a fragment of cloned DNA (the probe) is radiolabeled by using any of a variety of techniques. As the liquid perfuses the gel, it carries DNA fragments with it, depositing them on the membrane filter, to which the DNA sticks. Liquid is then forced through the agarose gel in a direction perpendicular to the direction in which the DNA moved during electrophoresis. Thus, after electrophoresis, a paper-thin membrane microfilter (made of nitrocellulose or nylon) is placed over the flat portion of the gel. The DNA fragments must be transferred to a solid support to which they are irreversibly bound in order to carry out nucleic acid hybridization studies. Because the agarose gel used in electrophoresis is thick and the DNA fragments can move within it, DNA in the gel is not in a suitable form for further analysis. The final goal of Southern blotting is to identify specific fragments of cut DNA by using nucleic acid hybridization. Digested DNA is arrayed by size using electrophoresis through a semisolid (more.) Genomic DNA is digested with a single restriction endonuclease resulting in a complex mixture of DNA fragments of different sizes, that is, molecular weights. At any time after electrophoresis begins, small molecules will be closer to the anode than will large molecules.) The agarose gel is usually cast in the form of a flat rectangle a few millimeters thick. The semiporous agarose allows molecules of DNA to pass with varying degrees of ease, at a rate inversely proportional to their size. (Because the phosphate groups in DNA make the molecules negatively charged, they will migrate toward the anode in an electric field. Electrophoresis through an agarose gel then separates these fragments according to size. Purified genomic DNA is digested with a specific restriction endonuclease, which, as described above, will produce an array of differently sized DNA fragments. The presence of these sequences usually means that the gene itself is present in the genomic DNA. 17 In general, it allows one to determine whether specific nucleotide sequences in a cloned probe are present in a sample of genomic DNA. The frequent concomitant finding of skin warts and oral HPV infection may suggest some kind of HPV-specific immunosuppression.One of the most useful techniques for analyzing a gene at the level of genomic DNA is Southern blotting, named for its originator, E.M. To conclude, oral HPV infections as detected by SBH and PCR are surprisingly common, but similar to the genital tract, the virus seems to exist in a latent form in the vast majority of cases. Hand warts were encountered significantly more frequently in patients with a concomitant oral HPV infection. Histology could not be relied on distinguishing HPV DNA positive and HPV DNA negative samples. The HPV types detected in the genital and oral mucosa of index patients differed in all except two cases. In clinically normal epithelium, 15.6% and 23.1% of the samples were HPV-positive with SBH and PCR, respectively. With SBH and PCR, 15.4% and 29.4% of the biopsies, respectively, contained HPV DNA. Furthermore, one hundred formalin-fixed, paraffin-embedded biopsies were analyzed by using polymerase chain reaction (PCR), combined with dot blot hybridization and biotinylated HPV DNA probes. The presence of human papillomavirus (HPV) in biopsies taken from clinically normal buccal mucosa (n = 212) and clinical lesions (n = 60) was examined by Southern blot hybridization (SBH) using 32P-labelled HPV DNA probes.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |