Compositions and methods for enhancing reverse transcriptase activity and/or reducing the inhibition of reverse transcriptase
Inventors
Meng, Xiangdong • GONG, Xiaosong • Wan, Cindy C T
Assignees
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Publication Number
US-12331338-B2
Publication Date
2025-06-17
Expiration Date
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.
Core Innovation
The invention provides a composition for reverse transcriptase (RT) reactions that enhances RT performance and reduces RT inhibition during RT-PCR and qPCR. The composition includes a buffer, dNTPs, an oligonucleotide dTn, potassium glutamate, poloxamer 188, polyvinyl sulfonic acid (PVSA), and one or more oligonucleotides selected from X1–Xn defined by randomized nucleotide patterns.
The composition further includes randomized oligonucleotides whose X1–X5, X1–X6, X1–X7, X1–X8, X1–X9, X1–X10, X1–X11, and X1–X12 are configured to be randomly ordered A and T nucleotides and randomly ordered G and C nucleotides, or configured such that at least one but less than a specified number are randomly selected from A and T while the remaining are randomly selected from G and C. This enables cDNA synthesis from RNA templates using randomized primers with low and/or high GC content.
The invention also addresses RT inhibition from heparin, formalin, and tannic acid by including potassium glutamate and poloxamer 188 in the composition. It further provides RT activity/yield and sensitivity improvements, including improved performance under low RNA input using PVSA, in addition to the randomized primer design and tolerance to RT inhibitors.
Claims Coverage
The provided claim set includes one independent composition claim with four inventive features. A separate independent method claim is not explicitly provided in the supplied material, although dependent claims indicate method-oriented RT efficiency and sensitivity improvements using an RNA template and reverse transcriptase.
Defined RT-enhancement and RT-inhibition-reduction composition
A composition comprising a buffer, dNTPs, oligonucleotide dTn (n selected from 5, 6, 7, 8, 9, 10, 11 or 12), potassium glutamate, poloxamer 188, polyvinyl sulfonic acid (PVSA), and one or more oligonucleotides selected from X1–Xn with specified randomized nucleotide ordering rules.
Overall, the independent claim coverage is directed to a defined reaction composition that combines specific buffer and nucleotide components, PVSA, potassium glutamate, and poloxamer 188, together with defined randomized oligonucleotides X1–Xn having controlled A/T versus G/C randomization rules. The partial claim set also references method improvements for RT efficiency and sensitivity, but those method claims are not fully provided as independent claim text in the supplied material.
Stated Advantages
Enhances reverse transcriptase (RT) activity/yield and sensitivity.
Improves RT tolerance in the presence of heparin.
Improves RT tolerance in the presence of formalin.
Improves RT tolerance in the presence of tannic acid.
Improves cDNA synthesis from RNA templates using randomized primers with low and/or high GC content.
Improves RT performance under low RNA input.
Documented Applications
Used for RT-PCR and qPCR with cDNA synthesis from RNA templates.
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