Investigating Whether NdeI-Cut Ends are Available to Incorporate Deoxynucleotides

Abstract

Numerous experiments have indicated that Nde I-digested fragments have low ligation efficiency. A proposed explanation for this behaviour was that the 3’ hydroxyl end is unavailable to incorporate additional nucleotides. That experiment labeled the ends of Nde Idigested fragments with biotin; however, the results raised the question of whether the procedure was size-dependent or if it provided support for the hypothesis. To try and answer the question, two ladders were created from λ DNA – one cleaved with Hind III and the other with Vsp I. Both ladders were subsequently labeled with biotin. pBR322 was also used to generate larger fragments if the process was indeed size-dependent. The plasmid was digested with both Hind III and Nde I, labeled with biotin, and then the fragments were digested again with Vsp I and Sal I before it was run on a 2% agarose gel. Western blot analysis was conducted after transferring segmented DNA bands to a nitrocellulose membrane by Southern blotting. The agarose gel showed that there were equal amounts of Nde I-digested fragments and Hind III-digested fragments; however, the IDV values indicated that there were more Hind III-digested fragments labeled with biotin than Nde I-cut segments. This pattern supports the hypothesis that Nde I-cut ends were unavailable to incorporate deoxynucleotides. Additionally, the autoradiograph film indicated that only the higher molecular weight bands of the two ladders were successfully labeled with biotin. As the lower molecular weight bands of the double digest were seen in the film, it may be possible that a limited amount of biotin was present during the labeling process. Another possibility for the absence of the lower bands on the film may be that the biotin had detached from the fragment ends during the Southern blot.