Whether recognition sites for particular restriction endonucleases are present in the gene. The degree of similarity between the chromosomal gene and the probe sequence.ģ. Whether a particular gene is present and how many copies are present in the genome of an organism.Ģ. We can get following information from the technique of Southern blotting ġ. Information Obtained from Southern Blotting : If the probe has been labelled with 32P, it will be radioactive, and the sheet will show a band of radioactivity where the probe is hybridized with the complementary fragment. Next, a probe consisting of purified, single-stranded DNA corresponding to a specific gene (or mRNA transcribed from that gene) is poured over the sheet.Īny fragment that has a nucleotide sequence complementary to the probe’s sequence will hybridize (base pair) with the probe. The double-stranded helix of each DNA fragment is then denatured into single strands by making the pH of gel basic, and the gel is “blotted” with a sheet of nitrocellulose, transferring some of the DNA strands to the sheet. In this procedure, called a Southern blot, DNA from the sample is cleaved into restriction fragments with a restriction endonuclease, and the fragments are spread apart by gel electrophoresis. If we want to detect the presence of a specific sequence in our mixed DNA sample then we will accordingly design the probe which will have complementary sequence to our target sequence. In this technique we exploit the property of a radio-labelled probe with the single stranded DNA. This technique was invented in 1975 by E.M. Southern blot hybridization detects target DNA fragments that have been size-fractionated by gel electrophoresis. doi: 10.The following points highlight the top seven blotting techniques used in detection of DNA. Dery KJ, Yean SL, Lin RJ (2008) Assembly and glycerol gradient isolation of yeast spliceosomes containing transcribed or synthetic U6 snRNA.Aksaas AK, Larsen AC, Rogne M, Rosendal K, Kvissel AK, Skalhegg BS (2011) G-patch domain and KOW motifs-containing protein, GPKOW a nuclear RNA-binding protein regulated by protein kinase A.Burd CG, Dreyfuss G (1994) RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing.Gaur RK, McLaughlin LW, Green MR (1997) Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron. Zillmann M, Zapp ML, Berget SM (1988) Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.Doroshenk KA, Tian L, Crofts AJ, Kumamaru T, Okita TW (2014) Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization.Calla-Choque JS, Figueroa-Angulo EE, Avila-Gonzalez L, Arroyo R (2014) Alpha-Actinin TvACTN3 of Trichomonas vaginalis is an RNA-binding protein that could participate in its posttranscriptional iron regulatory mechanism.Salinas T, El Farouk-Ameqrane S, Ubrig E, Sauter C, Duchene AM, Marechal-Drouard L (2014) Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs.Islam A, Schulz S, Afroz S, Babiuk LA, van Drunen Littel-van den Hurk S (2015) Interaction of VP8 with mRNAs of bovine herpesvirus-1.Zhao S, Xue Y, Hao J, Liang C (2015) The RNA-binding properties and domain of Rice stripe virus nucleocapsid protein.Zang S, Lin TY, Chen X, Gencheva M, Newo AN, Yang L, Rossi D, Hu J, Lin SB, Huang A, Lin RJ (2014) GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo.Ryder SP, Recht MI, Williamson JR (2008) Quantitative analysis of protein-RNA interactions by gel mobility shift.Rodriguez PL, Carrasco L (1994) Nonradioactive northwestern analysis using biotinylated riboprobes.Bagga PS, Wilusz J (1999) Northwestern screening of expression libraries.Qian Z, Wilusz J (1993) Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy.Bowen B, Steinberg J, Laemmli UK, Weintraub H (1980) The detection of DNA-binding proteins by protein blotting.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |