Simple modifications of the standard DNA sequencing protocol allow for sequencing through siRNA hairpins and other repeats.
RNAi is a relatively new but powerful technology used for monitoring gene silencing in many species. The RNA corresponding to the gene of interest can be delivered into cells using various protocols, but the approach where both sense and antisense strands are located in the same transcript seems to be the most promising. However, the DNA sequencing step is a challenge due to the formation of a strong hairpin structure. In this work we present a very simple modification to the standard DNA sequencing protocol that allows sequencing through such difficult regions. The modification does not require any additional enzymatic or chemical manipulations. It simply adds a 5-min heat-denaturation step at 98 degrees C, prior to addition of a Taq mix. The inclusion of this step is very effective in sequencing through many different categories of difficult templates. Other examples of the implementation of this step have been described previously and more detailed information on heat-denaturation of plasmid DNAs has been presented.
Kieleczawa
Jan
J
Biological Technologies Department, Wyeth Research, 87 Cambridgepark Dr., Cambridge, MA 02140, USA. jkieleczwa@wyeth.com
D034741:RNA, Small Interfering / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*
D034741:RNA, Small Interfering