tailieunhanh - Molecular Biology Problem Solver 27

Molecular Biology Problem Solver 27. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | lation sites. Binding protein sites have been engineered into the target DNA and degenerate sites containing the required restriction methylation sites have also been added Grimes Koob and Szybalski 1990 . However modifications in the recognition sequence of the binding protein can decrease the complex s half-life allowing unwanted methylation at the AC site. 2. Achilles Heel Cleavage-Triple Helix Formation. The second Achilles cleavage reaction uses oligonucleotide-directed triplehelix formation as a sequence specific DNA binding protein blocking agent Hanvey Schimizu and Wells 1990 Maher Wold and Dervan 1989 . Pyrimidine oligonucleotides bind to homopurine sites in duplex DNA to form a stable triple-helix structure. The blocking reaction is followed by methylation removal of the pyrimidine oligonucleotide and methylase and cleavage by the restriction endonuclease. Single-site cleavage has been demonstrated on yeast chromosomes by blocking with a 24 bp pyrimidine oligo Strobel and Dervan 1991a 1992 and on human chromosome 4 using a 16 bp oligo Strobel et al. 1991b . An advantage of this method over the DNA binding protein AC is the increase in frequency of sites. Insertion of the AC site into the genome is not required. Relatively short purine tracts can be targeted using sequence data. Degenerate probes can be used to screen for overlapping methylation restriction endonuclease sites when suitable sequence data are not available Strobel et al. 1991b . Reaction conditions for successful pyrimidine oligonucleotide AC are complex Strobel and Dervan 1992 . Triple helix formation using spermine can inhibit certain methylases or precipitate DNA in the low-salt reaction conditions required by some methy-lases. The narrow pH range for the protection reaction may not be compatible with conditions required for efficient methylation. Neutral or slightly acidic conditions promote highly stable triple helices but reduce sensitivity to single base mismatches Moser and Dervan .