Digital polymerase fidelity assay

    公开(公告)号:US10287620B2

    公开(公告)日:2019-05-14

    申请号:US15581401

    申请日:2017-04-28

    Abstract: Methods of determining polymerase fidelity are provided. In one embodiment, the method comprises filling a gapped plasmid with a polymerase to form a gap-filled plasmid, wherein the gap-filled plasmid comprises a gene encoding an protein that is functional or non-functional depending on the polymerase fidelity; forming a plurality of partitions from a solution comprising the gap-filled plasmid and a label for detecting the presence of the plasmid; detecting the presence of the gap-filled plasmid in one or more of the partitions; and determining the fidelity of the polymerase by determining a ratio of partitions containing the gene encoding a functional protein to partitions containing a gene encoding a non-functional protein.

    Compositions and methods for enhancing reverse transcriptase activity and/or reducing the inhibition of reverse transcriptase

    公开(公告)号:US11597920B2

    公开(公告)日:2023-03-07

    申请号:US16911421

    申请日:2020-06-25

    Abstract: The subject application pertains to compositions and methods for enhancing reverse transcriptase (RT) activity and/or reducing the inhibition of RT by inhibitors, such as formalin, tannic acid and/or heparin. In some embodiments, RT inhibition is reduced by the addition of potassium glutamate, histidine hydrochloride monohydrate, poloxamer 188, or any combination thereof to a reaction mixture comprising a polymerase. In other embodiments, RT is enhanced through the addition of a polyvinyl sulfonic acid sodium salt (PVSA) to a reaction mixture. The subject application also provides oligonucleotide primers for use in the reverse transcription of target sequences and its enhancement. These primers have high GC content or low GC content. Methods of using a RT inhibition reducer or a RT enhancer in a composition with an RNA template and RT improves RT yield, RT sensitivity, or RT tolerance to various chemicals are also provided.

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