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Figure 4. Comparison of ssDNA and ssRNA ladders by gel electrophoresis. (a) Agarose gel separation of ss100 DNA ladder and RiboRuler samples using denaturing (2 M formaldehyde) (top) or non-denaturing (bottom) 1.5% agarose gel electrophoresis. For these assays, and for those in (b) and (c), the first ten bands of the ss100 DNA ladder (100–1000 bases long) were purified from the larger rolling circle amplification (RCA) products by PAGE before use. (b) Denaturing (8 M urea) and (c) non-denaturing 8% PAGE separation of ss100 DNA ladder and RiboRuler samples. (d) Comparison of ss50 DNA ladder and ss100 DNA ladder by denaturing 8% PAGE. (e) Comparison of ss100 DNA ladder and ss200 DNA ladder by denaturing 8% PAGE.


RNA marker preparation RiboRuler (Fermentas Inc., Lafayette, CO, USA). Electrophoretic separation of the ss100 DNA ladder and RiboRuler nucleic acids in adjacent lanes of either denaturing or non-denaturing agarose gels revealed substantial differences in the mobilities of bands (Figure 4a). For example, the DNAs in the ss100 DNA ladder generated consistent banding patterns, whereas the RNAs in the RiboRuler sample exhibit some differences between denaturing and non-denaturing conditions, likely due to the formation of strong RNA structures by particular RNA sequences. Also, the DNA and RNA molecules of equal size do not co-migrate, which highlights the disadvan- tages of using RNA markers as surrogates


Vol. 54 | No. 6 | 2013


for ssDNA. Likewise, differences in gel mobility between these DNAs and RNAs are also observed when the two samples are separated by denaturing (Figure 4b) and non-denaturing (Figure 4c) PAGE. Te method used to produce the ss100


DNA ladder can be used to generate markers of any unit size increment simply by varying the number of nucleotides in the template DNA. For example, the addition of six nucleotides to the 44 nucleotides of the I-R3 deoxyribozyme complementary sequence yielded 50-nucleotide unit- length ssDNA products (ss50 DNA ladder) (Figure 4d) whereas the addition of 156 nucleotides yielded ssDNA markers with 200-nucleotide increments (ss200 DNA ladder) (Figure 4e).


342 Tus, our combined RCA/self-cleaving


deoxyribozyme scheme allows for the production of ssDNA markers with incre- ments of ~50 nucleotides or larger. Furthermore, ssDNA markers produced by this method can be easily internally- or 5’-radiolabeled using standard methods. For example, radiolabeling using γ-32P[ATP] and polynucleotide kinase can be carried out aſter removal of the 5’ phosphate group generated by deoxyribozyme hydrolysis. Tis makes possible the production of ssDNAs for use as markers that can overcome the problems of structure formation and altered mobility observed with some existing RNA markers. Moreover, since DNA is more stable than RNA, ssDNA markers will have a storage time that is far greater than that


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100ssDNA RiboRuler


TM


100ssDNA RiboRuler


TM


100ssDNA RiboRuler


TM


50ssDNA 100ssDNA


100ssDNA 200ssDNA


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