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Publication Number
US-12297493-B2
Publication Date
2025-05-13
Expiration Date
Abstract
The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules and cDNA and/or DNA libraries using modified reverse transcriptases.
Core Innovation
The invention relates to an R2 reverse transcription enzyme defined by an amino acid sequence selected from SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67. The enzyme is described in the context of generating complementary deoxyribonucleic acid molecules from template ribonucleic acid molecules, including low-abundance, low copy number, fragmented, and degraded templates.
The disclosed approach includes nucleic-acid amplification and RNA/cDNA library preparation, operating at substantially constant temperature and/or within a temperature range of from about 12°C to about 42°C, without thermal cycling. The description emphasizes low misincorporation error rate, low error rates during nucleic-acid synthesis, generation of complementary deoxyribonucleic acid molecules, and reduced nonspecific products.
Template jumping is described between a template ribonucleic acid molecule and an acceptor nucleic acid molecule, independent of sequence identity, and the workflow includes modified reverse transcriptase and non-LTR retrotransposon, including R2 variants. The document also describes downstream context involving cell-free DNA/circulating tumor DNA analysis and labeling, molecular barcoding with structured adaptors, and sequencing library performance improvements.
Claims Coverage
The claim coverage centers on a sequence-defined R2 reverse transcription enzyme and includes five additional inventive features: substantially constant temperature amplification, a defined 12°C to 42°C amplification range, a misincorporation error rate limit, a specific activity range for generating complementary DNA, and template jumping independent of sequence identity.
R2 reverse transcription enzyme defined by selected amino acid sequences
An R2 reverse transcription enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.
Amplification at substantially constant temperature
The R2 reverse transcription enzyme is configured to perform an amplification reaction at a substantially constant temperature.
Amplification at a defined temperature range
The R2 reverse transcription enzyme is configured to perform an amplification reaction at a temperature of from about 12°C to about 42°C.
Low misincorporation error rate
The R2 reverse transcription enzyme has a misincorporation error rate of at most about 1 base out of 100 bases.
High specific activity for generating complementary DNA
The R2 reverse transcription enzyme is configured to generate a plurality of complementary deoxyribonucleic acid molecules with a specific activity of from about 20,000 units/mg to about 140,000 units/mg.
Template jumping independent of sequence identity
The R2 reverse transcription enzyme is capable of template jumping independent of sequence identity between a template ribonucleic acid molecule and an acceptor nucleic acid molecule.
The claim coverage centers on a sequence-defined R2 reverse transcription enzyme, with additional inventive features emphasizing template jumping independent of sequence identity, low misincorporation error, quantified specific activity, and amplification at substantially constant temperature and/or within a defined temperature range.
Stated Advantages
Enhanced template jumping and increased overall activity are reported.
Low misincorporation error rate of at most about 1 base out of 100 bases.
Generates complementary deoxyribonucleic acid molecules with a specific activity of from about 20,000 units/mg to about 140,000 units/mg.
Reduces nonspecific products.
Allows faster single-vessel ("1-pot") workflows for cDNA/DNA libraries.
Supports low-input, fragmented, or degraded templates, including cell-free DNA and cell-free RNA.
Enables continuous complementary DNA synthesis.
Template jumping independent of sequence identity between a template ribonucleic acid molecule and an acceptor nucleic acid molecule.
R2 reverse-transcription template-jumping is described as efficient.
R2 reverse-transcription is described as less sensitive to acceptor-adapter sequences than template switching (e.g., MMLV).
The approach is described as enabling use of Illumina sequencing adapters with tested acceptors showing similar efficiency for RNA vs DNA acceptors.
The approach is described as insensitive to the acceptor 3′-end.
Poly-A tail length control is described as independent of time/enzyme concentration.
The cfDNA library process is described as retaining original strands for fidelity.
Barcode and index design tolerates amplification and sequencing errors.
Supports cell-free DNA/circulating tumor DNA analysis and labeling using PCR, digital PCR, qPCR, and sequencing with molecular barcoding.
Enables liquid biopsy mutation detection.
rRNA/tRNA depletion strategies are used to maximize sequencing throughput.
Documented Applications
Cell-free DNA/circulating tumor DNA analysis using PCR, digital PCR, qPCR, and sequencing.
Molecular barcoding for labeling in cfDNA/ctDNA analysis using structured adaptors, including Y-shaped, linear, and circular molecular barcoding adaptors, with a molecular barcode and sample index.
Analysis of template-jump products.
RNA/cDNA library preparation and nucleic-acid amplification in contexts involving circulating tumor DNA (ctDNA) and liquid biopsies, including RNA from single cells and tissues.
Processing of low copy number template and fragmented/degraded RNA/templates.
Cancer profiling/diagnosis/prognosis for cfDNA/cancer profiling using molecular barcoding and adaptors as described in the provided content.
Generation of cfDNA libraries, including circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA), using an R2-based two-step process with copy-strand removal (e.g., dUTP/UDG).
Illumina sequencing adapter usage via acceptors, supported by described efficiency comparisons for RNA vs DNA acceptors and insensitivity to acceptor 3′-end.
Control of poly-A tail length as part of R2-based workflows using ATP/dideoxy/un-extendable nucleotides.
rRNA/tRNA depletion to maximize sequencing throughput, including magnetic-bead hybrid capture and Cas9-guide RNA cleavage.
Kit components for nucleic acid workflows, including primers, dNTPs, polymerase, buffers, and sequencing reagents.
Liquid biopsy mutation detection.
Sequencing library performance improvements.
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