PMID:26500066

Citation	Song, Y, Liu, KJ and Wang, TH (2015) Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1. Sci Rep 5:15620
Abstract	MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences.
Links	PubMed PMC4620478 Online version:10.1038/srep15620
Keywords	Adenosine Triphosphate/metabolism; DNA/chemical synthesis; DNA/genetics; MicroRNAs/analysis; MicroRNAs/genetics; Oligonucleotides/chemical synthesis; Oligonucleotides/genetics; RNA/chemical synthesis; RNA/genetics; RNA Ligase (ATP)/genetics; Viral Proteins/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:RLIG		GO:0003910: DNA ligase (ATP) activity	ECO:0000314:		F		Figure 2 shows the catalysis results of T4 RNA ligase 1/T4 RNL1 in combination with varying levels of ATP in Enterobacteria phage T4.	complete CACAO 13469

References

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