DNA polymerase mutant suited to nucleic acid amplification from RNA
Inventors
Uemori, Takashi • Matsumoto, Hiroyuki • Saito, Kensuke • AKITOMO, Miwa
Assignees
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Publication Number
US-12018294-B2
Publication Date
2024-06-25
Expiration Date
Abstract
Provided are: a DNA polymerase mutant having reverse transcriptase activity, the DNA polymerase mutant including a sequence consisting of twelve specific amino acids A1-A12, wherein the DNA polymerase mutant having reverse transcriptase activity is characterized in that the A3 and/or A10 amino acid is substituted by a basic amino acid residue different from that prior to the introduction of mutation; a kit and a composition including the DNA polymerase; a method for producing the DNA polymerase; and a method for modifying an existing DNA polymerase having reverse transcriptase activity.
Core Innovation
The disclosed invention relates to a mutant of a wildtype DNA polymerase derived from Thermus thermophilus, where the wildtype DNA polymerase has reverse transcriptase activity. The mutant is defined by a conserved 12-amino-acid sequence comprising A1–A12 set forth in SEQ ID NO:18, while the remaining portion of the amino acid sequence retains high identity to the amino acid sequence set forth in SEQ ID NO:1. The key functional requirement is that the reverse transcriptase activity in the mutant is improved relative to the wildtype DNA polymerase.
The improved reverse transcriptase activity is achieved by substituting specific residues within the 12-amino-acid sequence. Either A3 is replaced by a basic amino acid residue, or both A3 and A10 are each replaced by a basic amino acid residue. The disclosure emphasizes that these motif substitutions within A1–A12 produce the improved RT performance while maintaining a largely wildtype-like sequence outside the motif.
The invention is further directed to embodiments used for reverse transcription and nucleic acid amplification performed in the same container, including one-step RT-PCR and RT-isothermal nucleic acid amplification. The disclosure reports reduced RT time and increased nucleic acid amplification performance, and it demonstrates detection in real samples such as norovirus stool. The document also describes related forms, such as kits and compositions, and discusses optional design variations including exonuclease-lacking variants and PCNA/PIP-box-related fusion formats.
Claims Coverage
The document’s independent claim includes a DNA polymerase mutant defined by a specific conserved 12-amino-acid A1–A12 motif with basic-residue substitutions at A3 and/or A10, while requiring high sequence identity to the wildtype outside that motif and improved reverse transcriptase activity. Dependent claims further constrain the sequence identity threshold outside A1–A12 and specify arginine as the basic residue, and they add kit and composition contexts.
Conserved A1–A12 motif in a Thermus thermophilus reverse-transcribing DNA polymerase mutant
A mutant of a wildtype DNA polymerase derived from Thermus thermophilus, where the wildtype DNA polymerase is the amino acid sequence set forth in SEQ ID NO:1 and comprises a sequence consisting of 12 amino acids A1–A12 set forth in SEQ ID NO:18; the mutant comprises a sequence consisting of the 12 amino acids A1–A12.
Basic-residue substitutions at A3 and/or A10 within the A1–A12 motif
The mutant has either (i) A3 replaced by a basic amino acid residue, or (ii) A3 and A10 each replaced by a basic amino acid residue, while retaining the 12-amino-acid A1–A12 sequence.
High identity to the wildtype outside the A1–A12 motif with improved RT activity
In the portion outside the 12-amino-acid sequence of A1–A12, the mutant has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1, and the reverse transcriptase activity in the mutant is improved relative to the wildtype DNA polymerase.
Tightened outside-motif identity threshold
The mutant has at least 95% amino acid sequence identity in the portion outside the A1–A12 12-amino-acid sequence relative to the amino acid sequence set forth in SEQ ID NO:1.
Arginine-specific substitutions at A3 and/or A10
Either residue A3 is replaced by arginine or both residues A3 and A10 are each replaced by arginine in the mutant.
Kit containing the claimed mutant
A kit that includes the mutant described in claim 1.
Composition comprising the claimed mutant
A composition that comprises the mutant described in claim 1.
Overall, the claim set focuses on a Thermus thermophilus DNA polymerase mutant that preserves the A1–A12 motif (SEQ ID NO:18) while introducing basic-residue substitutions at A3 and/or A10, maintaining high sequence identity outside the motif, and improving reverse transcriptase activity; the dependent claims further restrict sequence identity and define arginine substitutions and provide kit/composition embodiments.
Stated Advantages
Reduced RT time.
Increased nucleic acid amplification performance, including higher PCR starting template/cDNA yield (e.g., reduced Ct values).
Enhanced detection in real samples, including norovirus stool.
Documented Applications
One-step RT-PCR and RT-isothermal nucleic acid amplification performed in the same container.
Detection in real samples including norovirus stool.
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