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.
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.
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