The staining efficiency of cyanine dyes for single‐stranded DNA is enormously dependent on nucleotide composition

Abstract

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson‐Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.

Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

“…The selectivity and the yield for the formation of DNA rings were calculated referring to the method of our previous papers (see Experimental Section) . Rather poor staining of 50‐nt 2L α‐50 in lane 1 of Figure A is ascribed to its GA rich sequence …”

“…[4,5] Rather poor staining of 50-nt2L a-50 in lane 1o fF igure 1A is ascribed to its GA rich sequence. [8] Figure 2. Preparation of 148-nt c-ssDNA by one-pot ligation of 3L a'-50 ,3 L b-39, and 3L g-59 (50-, 39-, and 59-nt).…”

“…Polymeric byproducts were hardly observable. By removing the l-ssDNA by exonuclease digestion, the 200-nt c-ssDNAw as obtained in almostcompletelyp ure form (lanes [6][7][8].…”

Co-transcriptional RNA strand displacement circuits

“…Lane 5: separate transcription of the output (O2) and gate′ strands. The 46 base single-stranded O2 strand stained poorly for visualization(53). See Supplementary Fig.2for control transcript designs.…”

“…Lane 3 to lane 7: 25 nmol/L of 1_2 gate template was co-transcribed with 2.5 nmol/L (0.1x) to 50 nmol/L (2x) of I1 template. The 46 base output strand of the gate (O2) was not visible(53). Electrophoresis was conducted 2 h DNase I addition.…”