Life Sciences

Life Sciences

CirclSeq - Automatable and relibale preparation of a DNA library in a one-vial reaction for unbiased quantification of mRNA

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Angebotsbeschreibung

Ref.-Nr. 4031

The analysis of the mRNA content of a cell or a tissue via sequencing provides a method for functional analysis. In common protocols, prior to the sequencing procedure itself the mRNA has to be reverse transcribed into cDNA, followed by random shearing into cDNA fragments, linker ligation, and amplification via PCR. The library of PCR amplicons can then be sequenced by various methods of next generation sequencing (NGS).

In many protocols, the primers used for the reverse transcription (RT) or ligation have to be removed before sequencing. Typically, this is achieved by performing a polyacrylamide gel electrophoresis, which suffers from poor quantitative yield and poor discrimination between molecules of similar size. Additionally, PCR amplification can lead to biased quantification of rare mRNA species.

The present invention allows overcoming these problems by using a new protocol, which consists of the following steps:

1) RT of mRNA into cDNA in presence of dUTP
2) RNA digestion and 3’ end blocking of RT primer with ddTTP
3) Enzymatic cleavage at positions of dUTP incorporation 
4) cDNA circularization 
5) NGS

The blocking of all 3' ends by the untemplated incorporation of di-desoxy-TTP (ddTTP) by terminal desoxynucleotidyl transferase hinders non-elongated primers from circularization and thus from later sequencing. Additional purification steps to remove the primers are not necessary anymore. cDNA digestion at dUTP sites still gives rise to 3'OH ends that can be circularized and analyzed by deep sequencing subsequently.

Thus, this novel method allows much higher throughput and maximizes the overall yield and the fraction of sequencing reads corresponding to real mRNA templates.

Commercial Opportunities

This method offers the possibility of an automatable, time- and cost-saving high throughput sequencing technique of mRNA or other nucleic acid molecules without the need for prior amplification.

On behalf of the University Bonn, PROvendis offers access to rights for commercial use as well as the opportunity for further co-development.

Current Status 

In case of interest we are pleased to inform you about the current patent status.


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