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使用SplintR连接酶的灵敏且特异的miRNA检测方法。

Sensitive and specific miRNA detection method using SplintR Ligase.

作者信息

Jin Jingmin, Vaud Sophie, Zhelkovsky Alexander M, Posfai Janos, McReynolds Larry A

机构信息

Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA.

Division of RNA Biology, New England Biolabs, Ipswich, MA 01938-2773, USA

出版信息

Nucleic Acids Res. 2016 Jul 27;44(13):e116. doi: 10.1093/nar/gkw399. Epub 2016 May 6.

DOI:10.1093/nar/gkw399
PMID:27154271
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5291259/
Abstract

We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.

摘要

我们描述了一种简单、特异且灵敏的微小RNA（miRNA）检测方法，该方法利用小球藻病毒DNA连接酶（SplintR®连接酶）。这种两步法包括将与miRNA杂交的相邻DNA寡核苷酸连接起来，然后进行实时定量PCR（qPCR）。对于RNA夹板式DNA连接，SplintR连接酶比T4 DNA连接酶或T4 RNA连接酶2快100倍。SplintR连接酶高效连接时，DNA探针与miRNA之间只需4-6个碱基对的重叠。与使用逆转录酶进行miRNA cDNA合成的方法相比，这一特性在设计miRNA特异性连接探针时具有更大的灵活性。qPCR SplintR连接测定法很灵敏；它能检测到几千个miR-122分子。对于miR-122检测，使用FAM标记的双淬灭DNA探针的SplintR qPCR测定法比TaqMan测定法至少灵敏40倍。当与新一代测序相结合时，SplintR方法可对来自脑、肾、睾丸和肝脏的miRNA进行多重检测。SplintR qPCR测定法具有特异性；能检测出相差一个核苷酸的单个let-7 miRNA。SplintR连接酶快速的动力学特性以及连接与具有短互补序列的RNA杂交的DNA探针的能力，使其成为一种用于miRNA检测的有用酶。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/c1f66a53b1eb/gkw399fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/bc6290d1ae47/gkw399fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/09fdb82ca583/gkw399fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/d818132b80f7/gkw399fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/d426e2ed4798/gkw399fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/32dcca8f0864/gkw399fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/b8fda3158d8a/gkw399fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/c1f66a53b1eb/gkw399fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/bc6290d1ae47/gkw399fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/09fdb82ca583/gkw399fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/d818132b80f7/gkw399fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/d426e2ed4798/gkw399fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/32dcca8f0864/gkw399fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/b8fda3158d8a/gkw399fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/119e/5291259/c1f66a53b1eb/gkw399fig7.jpg

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