Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.
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.
Biomedical Engineering Department, Johns Hopkins University, Baltimore, MD, 21218, USA.
Yunke
Y
Song
Mechanical Engineering Department, Johns Hopkins University, Baltimore, MD, 21218, USA.|Circulomics Inc, Baltimore, MD, 21211, USA.
Kelvin J
KJ
Liu
Biomedical Engineering Department, Johns Hopkins University, Baltimore, MD, 21218, USA.|Mechanical Engineering Department, Johns Hopkins University, Baltimore, MD, 21218, USA.
Tza-Huei
TH
Wang
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D012313:RNA / Q000138:chemical synthesis* / Q000235:genetics; D011130:RNA Ligase (ATP) / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D035683:MicroRNAs; D009841:Oligonucleotides; D014764:Viral Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1