| Popis: |
Publisher Summary Electrophoretic migration of RNA is affected by its terminal OH or P character to a sizable and variable extent, considerably more than is DNA. If not accounted for when sizing RNA, errors of up to two nucleotides can arise, even when using sequencing ladders prepared from RNA with the same sequence. Many cellular cleavage events of interest generate 3′-OH termini, yet these termini are not formed when using the standard methods of preparing RNA sequencing ladders, which all generate 2′,3′-cyclic P and 5′-OH termini. Fortunately, conditions have been found in which nuclease P1 cleaves in a highly G-specific manner and different conditions in which it cleaves in an A-preferential manner, allowing convenient preparation of the desired sequencing ladders. The key to accurate RNA size determination is to select appropriate marker ladders that not only are from the same sequence RNA but also have comparable termini. Cleavage agents commonly used to generate such ladders are sequence-specific RNases, including RNase T1 (which cleaves after G residues) and RNase A (which cleaves after C and U residues), and alkali, which cleaves after any residue. |
