Granted Patent
B2
US 12,297,493 · App. 17/461,348 · Granted May 13, 2025
Methods for processing nucleic acid samples
Assignee:
Bio-Rad Laboratories, Inc.
C12Q1/686C12N9/1276C12N15/1096C12P19/34C12Q1/68C12Q1/6806C12Q1/6846C12Q2600/112C12Q2600/118C12Q2600/158C12Q2600/16C12Q2600/178C12Y207/07049
View Patent ↗
Loading inventors, assignments & file history…
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.
Claims (25)
1. 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.
2. The R2 reverse transcription enzyme of claim 1 , configured to perform an amplification reaction at a substantially constant temperature.
3. The R2 reverse transcription enzyme of claim 1 , configured to perform an amplification reaction at a temperature of from about 12° C. to about 42° C.
4. The R2 reverse transcription enzyme of claim 1 , having a misincorporation error rate of at most about 1 base out of 100 bases.
5. The R2 reverse transcription enzyme of claim 1 , having a misincorporation error rate of at most about 1 base out of 10,000 bases.
6. The R2 reverse transcription enzyme of claim 1 , configured to generate a plurality of complementary deoxyribonucleic acid molecules from one or more template ribonucleic acid molecules with a specific activity of from about 20,000 units/mg to about 140,000 units/mg.
7. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 49.
8. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 50.
9. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 51.
10. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 53.
11. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 54.
12. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 55.
13. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 56.
14. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 57.
15. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 58.
16. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 59.
17. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 60.
18. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 61.
19. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 62.
20. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 63.
21. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 64.
22. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 65.
23. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 66.
24. The R2 reverse transcription enzyme of claim 1 , comprising the amino acid sequence of SEQ ID NO: 67.
25. The R2 reverse transcription enzyme of claim 1 , capable of template jumping independent of sequence identity between a template ribonucleic acid molecule and an acceptor nucleic acid molecule.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 31, 2023
From: BIBILLO, ARKADIUSZ; PATEL, PRANAV
To: 2D GENOMICS INC.
Reel/Frame 063803/0813 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 31, 2023
From: 2D GENOMICS INC.
To: BIO-RAD LABORATORIES, INC.
Reel/Frame 063803/0884 →
Continuity (5)
Continuation
PCTUS2017061197
· Nov 10, 2017
Provisional Application
62477211
· Mar 27, 2017
Provisional Application
62421028
· Nov 11, 2016
References Cited (138)
US 4663290A
· Weis et al.
· 1987
[cited by applicant]
US 4889818A
· Gelfand et al.
· 1989
[cited by applicant]
US 4965188A
· Mullis et al.
· 1990
[cited by applicant]
US 5374553A
· Gelfand et al.
· 1994
[cited by applicant]
US 5605793A
· Stemmer
· 1997
[cited by applicant]
US 5811238A
· Stemmer et al.
· 1998
[cited by applicant]
US 5830721A
· Stemmer et al.
· 1998
[cited by applicant]
US 5834252A
· Stemmer et al.
· 1998
[cited by applicant]
US 5837458A
· Minshull et al.
· 1998
[cited by applicant]
US 5962272A
· Chenchik et al.
· 1999
[cited by applicant]
US 5972603A
· Bedford et al.
· 1999
[cited by applicant]
US 6444468B1
· Stemmer et al.
· 2002
[cited by applicant]
US 7153672B1
· Eickbush et al.
· 2006
[cited by applicant]
US 7229765B2
· Ziman et al.
· 2007
[cited by applicant]
US 7910354B2
· Drmanac et al.
· 2011
[cited by applicant]
US 8518640B2
· Drmanac et al.
· 2013
[cited by applicant]
US 9127302B2
· Verrant et al.
· 2015
[cited by applicant]
US 9228228B2
· Drmanac et al.
· 2016
[cited by applicant]
US 9328382B2
· Drmanac et al.
· 2016
[cited by applicant]
US 9410173B2
· Betts et al.
· 2016
[cited by applicant]
US 9546389B2
· Li
· 2017
[cited by applicant]
US 9617581B2
· Keene et al.
· 2017
[cited by applicant]
US 9708654B2
· Hunicke-Smith et al.
· 2017
[cited by applicant]
US 20040137588A1
· Scrofani et al.
· 2004
[cited by applicant]
US 20060281079A1
· Eickbush et al.
· 2006
[cited by applicant]
US 20070111944A1
· Scrofani et al.
· 2007
[cited by applicant]
US 20070255053A1
· Ziman et al.
· 2007
[cited by applicant]
US 20080268508A1
· Sowlay
· 2008
[cited by applicant]
US 20100022414A1
· Link et al.
· 2010
[cited by applicant]
US 20100297728A1
· Ziman et al.
· 2010
[cited by applicant]
US 20110206638A1
· Choi
· 2011
[cited by applicant]
US 20120208199A1
· Ziman et al.
· 2012
[cited by applicant]
US 20130344491A1
· Ziman et al.
· 2013
[cited by applicant]
US 20140057799A1
· Johnson et al.
· 2014
[cited by applicant]
US 20140147851A1
· Qian
· 2014
[cited by applicant]
US 20140302504A1
· Belhocine et al.
· 2014
[cited by applicant]
US 20150191781A1
· Ziman et al.
· 2015
[cited by applicant]
US 20160194686A1
· Drmanac et al.
· 2016
[cited by applicant]
US 20170327866A1
· Keene et al.
· 2017
[cited by applicant]
US 20170369932A1
· Qian
· 2017
[cited by applicant]
US 20180002735A1
· Drmanac et al.
· 2018
[cited by applicant]
US 20180044726A1
· Hunicke-Smith et al.
· 2018
[cited by applicant]
US 20200277664A1
· Frenz
· 2020
[cited by applicant]
EP 2820158A1
· 2015
[cited by applicant]
WO 9610640A1
· 1996
[cited by applicant]
WO 2015126927A2
· 2015
[cited by applicant]
WO 2018089860A1
· 2018
[cited by applicant]
S. M. Christensen et al, “Role of the Bombyx mori R2 element N-terminal domain in the target-primed reverse transcription (TPRT) reaction”, Nucleic Acids Research, GB, (20051127), vol. 33, No. 20, doi:10.1093/nar/gki957…
[cited by applicant]
Varuni K. Jamburuthugoda et al, “Identification of RNA binding motifs in the R2 retrotransposon-encoded reverse transcriptase”, Nucleic Acids Research, (20140623), vol. 42, No. 13, doi:10.1093/nar/gku514, ISSN 0305-1048…
[cited by applicant]
Hou E W et al, “High-level expression and purification of untagged and histidine-tagged HIV-1 reverse transcriptase”, Protein Expression and Purification, Academic Press, San Diego, CA, (20040301), vol. 34, No. 1, doi:1…
[cited by applicant]
Anisimova , 2008, Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab, BMC Biochem 9:14.
[cited by applicant]
Bibillo , 2002, High Processivity of the Reverse Transcriptase from a Non-long Terminal Repeat Retrotransposon, J Biol Chem 277(38):34836.
[cited by applicant]
Don , 1993, A “one tube reaction” for synthesis and amplification of total cDNA from small numbers of cells, Nucleic Acids Research 21(3):783.
[cited by applicant]
Fujimoto , 2004, Integration of the 5′ end of the retrotransposon, R2Bm, can be complemented by homologous recombination, Nucleic Acids Res 32:1555-1565.
[cited by applicant]
Gu , 2016, Depletion of Abundant Sequences by hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications, Genome Biol 17:41.
[cited by applicant]
Islam , 2011, Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq, Genome Res 21:1160-1167.
[cited by applicant]
Jamburuthugoda , 2011, The reverse transcriptase encoded by the non-LTR retrotransposon R2 is as error prone as that encoded by HIV-1, J Mol Biol 407:661-672.
[cited by applicant]
Kannan , 2016, One step engineering of the small subunit ribosomal RNA using CRISPR/Cas9, Sci Rep 6:1-10.
[cited by applicant]
Kurzynska , 2007, DNA-directed DNA polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon, J Mol Biol 374:322-333.
[cited by applicant]
Schmidt , 1999, CapSelect: A highly sensitive method for 5′ CAPdependent enrichment of full-length cDNA in PCR mediated analysis of mRNAs, Nucl Acids Res 21(3):783.
[cited by applicant]
Schramm , 2000, A simple and reliable 5′-RACE approach, Nucl Acids Res 28(22):e96.
[cited by applicant]
Song , 2020, Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing, Microbiome 8:80.
[cited by applicant]
Tani , 2007, Circulating Cell-free mRNA in Plasma as a Tumor Marker for Patients with Primary and Recurrent Gastric Cancer, Anticanc Res 27:1207-1212.
[cited by applicant]
Zhulidov , 2004, Simple cDNA normalization using kamchatka crab duplex-specific nuclease, Nucleic Acids Res 32(3):e37.
[cited by applicant]
AB076841: Bombyx mori non-LTR retrotransposon R2Bm gene for reverse transcriptase, complete cds and 28S rRNA.
[cited by applicant]
Anisimova Et al., Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab, BMC Biochemistry 9:14.
[cited by applicant]
Benes, 2011, Ribo-Zero Gold kit: improved RNA-seq results after removal of cytoplasmic and mitochondrial ribosomal RNA., Nature Methods, vol. 8, pp. iii-iv.
[cited by applicant]
Bibilo et al., End-to-ed template jumping by the reverse transcriptase encoded by the R2 retrotransposon, J Biol Chem, Apr. 9, 2004;279(15):1494-53.
[cited by applicant]
Bibillo et al., High Processivity of the Reverse Transcriptase from a Non-long Terminal Repeat Retrotransposon, J. of Biological Chemistry, 2002, 277(38):34836-45.
[cited by applicant]
Burke, et al., R4, a non_LTR retrotransposon specific to the large subunit rRNA genes of nematodes. Nucleic Acids Res. Nov. 25, 1995, 23(22):4628-4634.
[cited by applicant]
Co-pending U.S. Appl. No. 16/167,030, filed Oct. 22, 2018.
[cited by applicant]
Co-pending U.S. Appl. No. 16/168,002, filed Oct. 23, 2018.
[cited by applicant]
Cordaux et al., The impact of retrotransposons on human genome evolution. Nature Reviews Genetics, Oct. 2009; 10:691-703.
[cited by applicant]
Datsenko et al., One-step inactivation of chromosomal genes in
[cited by applicant]
Don et al., A “one tube reaction” for synthesis and amplification of total cDNA from small numbers of cells, Nucleic Acids Research, 1993, 21(3):783.
[cited by applicant]
Fujimoto et al., Integration of the 5′ end of the retrotransponson, R2Bm, can be complemented by homologous recombination, Nucleic Acids Res, Mar. 3, 2004, vol. 32, pp. 1555-1565.
[cited by applicant]
Fungtammasan, 2016, Reverse Transcription Errors and RNA-DNA Differences at Short Tandem Repeats, Mol Biol Evol, vol. 33, No. 10, 2744-2758.
[cited by applicant]
Gonzalez-Roca et al., Accurate Expression Profiling of Very Small Cell Populations, PLOS ONE, 2010; 5(12):e14418.
[cited by applicant]
Gu et al., Depletion of Abundant Sequences by hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications, Genome Biology, 2016, 17:41.
[cited by applicant]
Hashimshony et al., CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification, Cell Rep. Sep. 27, 2012; 2(3):666-73.
[cited by applicant]
Hizi et al., RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzme, J. Biol. Chem. Apr. 10, 1997; 252(17):2281-2289.
[cited by applicant]
International Search Report and Written Opinion issued in PCT/US2017/061197, date of mailing: Feb. 23, 2018.
[cited by applicant]
Islam et al., Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq., Genome Research, 2011, 21:1160-1167.
[cited by applicant]
Jamburuthugoda, 2011, The reverse transciptase encoded by the non-LTR retrotransponson R2 is as error prone as that encoded by HIV-1, J Mol Biol, 407(5):661-72.
[cited by applicant]
Kanamori-Katayama et al., Unamplified cap analysis of gene expression on a single-molecule sequencer, Genome Re., Jul. 2011:21(7):1150-1159.
[cited by applicant]
Kannan et al., One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9, Scientific Reports,, 2016, 6:3074, 10 pages.
[cited by applicant]
Kapteyn, 2010, Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and omproves cDNA synthesis from very small RNA samples. BMC Genomics, vol. 11, 9 pages.
[cited by applicant]
Kurimoto et al., An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res. Mar. 17, 2006; 34(5):e42.
[cited by applicant]
Kurzynska-Kokorniak, 2007, DNA-directed DNA polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon, J Mol Biol, 374(2):322-333.
[cited by applicant]
Le Grice et al., Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse tramscriptase, EMBO Journal, Dec. 1991;10(12):3905-3911.
[cited by applicant]
Lorenzo et al., PCR-based method for the introduction of the mutations in genes cloned and expressed in vaccinia virus, Biotechniques, Feb. 1998;24(2):308-13.
[cited by applicant]
Lutz et al., Creating multiple-crossover DNA libraries independent of sequence identity, Proc Natl Acad Dci USA, Sep. 25, 2011; 98(20):11248-53.
[cited by applicant]
Ngo et al., 1994 Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox, The Protein Folding Problem and Tertiary Structure Prediction, Merz et al. (ed.), Birkhauser, Boston, MA pp. 433 and 49…
[cited by applicant]
O'Neil et al., Ribosomal RNA Depletion for Efficient Unitfor Efficient Use of RNA-Seq Capacity, Current Protocols in Molecular Biology, 2013, 4.19.1-4.19.8.
[cited by applicant]
Ozsolak et al., Digital transcriptome profiling from attomole-level RNA samples. Genome Res. Apr. 2010;20(4):519-25.
[cited by applicant]
Picelli, 2014, Full-length RNA-seq from single cells using Smart-seq2., Nature Protoc, vol. 9, No. 1, pp. 171-181.
[cited by applicant]
Qiu et al., Single-neuron RNA-Seq: technical feasibility and reproducibility, Front Genet, Jul. 6, 2012;3:124.
[cited by applicant]
Saiki et al., Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science, Jan. 29, 1988; 239(4839):487-91.
[cited by applicant]
Scherrer et al., Historical review: The discovery of ‘giant’ RNA and RNA processing: 40-years of enigma, Trends in Biomedical Sciences, 2003, 28(10):566.
[cited by applicant]
Schmidt et al., CapSelect: A highly sensitive method for 5′ CAPdependent enrichment of full-length cDNA in PCR mediated analysis of mRNAs, Nucleic Acids Research, 1999, 21(3):783.
[cited by applicant]
Schramm et al., A simple and reliable 5′-RACE approach, Nucleic Acids Research, 2000, 21(3):783.
[cited by applicant]
Simon et al., A diversity of uncharacterized reverse transcriptases in bacteria, Nucleic Acids Res., Dec. 2008; 36(22):7219-7229.
[cited by applicant]
Song et al., Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing, Microbiome, 2020, 8:80.
[cited by applicant]
Superscript II Reverse Transcriptase, Product Description download Oct. 2020.
[cited by applicant]
Tang et al., mRNA-Seq whole-transcriptome analysis of a single cell, Nature Methods, 2009, 6(5):377.
[cited by applicant]
Tani et al., Circulating Cell-free mRNA in Plasma as a Tumor Marker for Patients with Primary and Recurrent Gastric Cancer, Anticancer Research, 2007, 27:1207-1212.
[cited by applicant]
Temin, 1985, Reverse transcription in the eukaryotic genome: retroviruses, pararetroviruses, retrotransposons, and retrotranscripts, Molecular Biology and Evolution, Nov. 1, 1985; 2(6):455-468.
[cited by applicant]
Werner et al., Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase aβ: Localization of the Polymerase and RNase H Active Sites in the a Subunit, J Virol. Apr. 2000; 74(7):3245-3252.
[cited by applicant]
Werner et al., Soluble Rous Sarcoma Virus Reverse Transcriptasea, aβ, and β Purified from Insect Cells Are Processive DNA Polymerases That Lack an RNase H 3′ ® 5′ Directed Processing Activity, J. Biol. Chem. 1999; 274(3…
[cited by applicant]
Written Opinion issued in Singapore Patent Application No. 11201904202S, date of mailing: Sep. 21, 2020, 11 pages.
[cited by applicant]
Xiong et al., Origin and evolution of retroelements based upon their reverse transcriptase sequences, EMBO J. Oct. 1990;9(10):3353-62.
[cited by applicant]
Zhou et al., Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye, Clin Chem, Aug. 2004;50(8):1328-35.
[cited by applicant]
Zhulidov et al., Simple cDNA normalization using kamchatka crab duplex-specific nuclease, Nucleic Acids Research, 2004, 32(3):e37.
[cited by applicant]
Zoller et al., Oligonucleotide-drected mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA, Nucl. Acids Res., Oct. 25, 1982; 10(20):6487…
[cited by applicant]