Two-tailed PCR

Analysis of short nucleic acids and direct blood genotyping.

In 2017 TATAA Biocenter and the Institute of Biotechnology, BIOCEV, developed Two-tailed PCR for ultrasensitive analysis of microRNAs.

This is a highly specific, sensitive and cost-effective system is used to quantify miRNA expression based on two-step RT-qPCR, with SYBR-green detection chemistry. The results of this work can be found in the prestigious journal Nucleic Acids Research.

Two-tailed PCR
Schematic of Two-tailed RT-qPCR. (A) Two-tailed RT primer having two hemiprobes connected by a hairpin folding sequence. (B) The hemiprobes bind cooperatively, one at each end of the target miRNA, forming a stable complex. (C) Reverse transcriptase binds the 3′-end of the hybridized Two-tailed RT primer and elongates it to form tailed cDNA. (D) The cDNA is amplified by qPCR using two target-specific primers.

Mature microRNAs are single-stranded molecules only 22-24 nucleotides in length. They constitute a recently discovered class of non-coding RNAs that plays a key role in the regulation of gene expression. Many microRNAs are present in families that differ only in a single base position or terminal modification. Acting at the post-transcriptional level, microRNAs fine-tune the expression of many protein-encoding genes. microRNAs are present in tissues, but also in biofluids, where they are actively transported as reporter molecules. This feature makes microRNAs exceedingly attractive biomarkers and its use is currently being standardized by the European effort SPIDIA.

There are several techniques to measure microRNA levels with quantitative PCR, but current methods use conventional pairs of primers, each 20-25 bases in length. To be able to amplify the short microRNAs they must be elongated, which often compromises sensitivity and specificity. The novel Two-tailed approach solves this problem by sensing the microRNA sequencing with two short hemiprobes. Neither hemiprobe is long enough to bind alone, but in the novel Two-tailed design the hemiprobes are connected, which leads to cooperative binding, such that either probes bind, which gives sufficient contact and stability, or neither binds. Since each hemiprobes is short, a mismatch in its binding site has a major impact, leading to the enhanced specificity of the Two-tailed method.

Two-tailed PCR was compared to three other methods for microRNA detection is found to be more sensitive and specific, being capable to detect fewer than 10 microRNA molecules in a sample. Two-tailed PCR can be multiplexed to essentially any degree using 2-tube protocol and when using one-tube protocol multiplexing with probes is possible. Two-tailed PCR is one of the methods currently evaluated for microRNA detection in liquid biopsies collected for the management of cancer within the European effort Cancer-ID and for Sepsis within the European effort Smartdiagnos.

Custom Two-tailed assays can be ordered here.

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