2.2.3 Prevention of methylation site specific adapters’ self-ligation
The enzymatic products with methylation dependent restriction endonucleases yield highly viscous end with four-base extrusion. To use these enzymatic products for gnomic sequencing, adapters with four wobble bases of NNNN is a reasonable option. To minimize or eliminate the potential self-ligation of the NNNN adapters, the YNNY and RNNR adapters were compared with NNNN adapters in the condition of T4 ligase. The ligation reaction was set in conditions as shown in the Table 4. After ligation reaction completed, 5 μL of each ligation reactions was visualized by agarose gel electrophoresis under 100 V for 30 min.
Results:
3.1 CGat adapters’ self-ligation prevented by enzyme ClaI
As shown in Fig. 2, with T4 DNA ligase, CAat adapters were completely self-ligated (lane 2). The addition of restriction enzyme ClaI efficiently prevented the formation of self-ligated products. Fig. 3 illustrated the strategy of preventing CGat adapters’ self-ligation and promoting adapter-insert ligation. Lanes 1 and 2 show the artificial template and its enzymatic product by HpaII digestion. Without ClaI, both adapters’ self-ligated products and the products from insert and adapters ligation were observable, but adapters’ self-ligated products are predominant (lane 4). Three observable bands in lane 5 were the un-ligated adapters, self-ligated adapters, and the ligation products of insert and adapters, in which the ligation products of insert and adapters are predominant instead. The ligation products of insert and adapters were confirmed by Sanger sequencing, with characteristic nucleotides CGAT in the sense strand (Fig.3 b).