Recombinant polymerases for incorporation of protein shield nucleotide analogs
First Claim
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1. A method of sequencing a DNA template, the method comprising:
- a) providing a reaction mixture comprising;
the DNA template,a replication initiating moiety that complexes with or is integral to the template,at least one nucleotide analog, which analog comprises a polypeptide moiety positioned between a dye moiety and a nucleotide component of the analog, wherein the polypeptide moiety comprises at least 60 amino acids; and
a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 or at least 90% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position I524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction;
b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA; and
c) identifying a time sequence of incorporation of the at least one nucleotide analog into the resulting DNA.
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Abstract
Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing or nucleic acid amplification. Such properties include enhanced performance with large nucleotide analogs, increased stability, increased readlength, and improved detection of modified bases, and can also include resistance to photodamage, enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased accuracy, altered speed, increased cosolvent resistance, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.
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Citations
24 Claims
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1. A method of sequencing a DNA template, the method comprising:
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a) providing a reaction mixture comprising; the DNA template, a replication initiating moiety that complexes with or is integral to the template, at least one nucleotide analog, which analog comprises a polypeptide moiety positioned between a dye moiety and a nucleotide component of the analog, wherein the polypeptide moiety comprises at least 60 amino acids; and a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 or at least 90% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position I524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction;b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA; and c) identifying a time sequence of incorporation of the at least one nucleotide analog into the resulting DNA. - View Dependent Claims (2, 6, 7, 8, 9)
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3. A method of making a DNA, the method comprising:
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(a) providing a reaction mixture comprising; a template, a replication initiating moiety that complexes with or is integral to the template, at least one nucleotide analog, which analog comprises a polypeptide moiety positioned between a dye moiety and a nucleotide component of the analog, wherein the polypeptide moiety comprises at least 60 amino acids; and a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 or at least 90% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position 1524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction; and(b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA. - View Dependent Claims (4, 5, 10, 11, 12, 13, 14)
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15. A method of sequencing a DNA template, the method comprising:
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a) providing a reaction mixture comprising; the DNA template, a replication initiating moiety that complexes with or is integral to the template, at least one nucleotide analog comprising a fluorescent label, which analog has a molecular weight of at least 10,000; and a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 or at least 90% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position I524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction;b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA; and c) identifying a time sequence of incorporation of the at least one nucleotide analog into the resulting DNA. - View Dependent Claims (16, 17, 18)
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19. A method of making a DNA, the method comprising:
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(a) providing a reaction mixture comprising; a template, a replication initiating moiety that complexes with or is integral to the template, at least one nucleotide analog comprising a fluorescent label, which analog has a molecular weight of at least 10,000; and a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;
1 or at least 90% identical to SEQ ID NO;
2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position 1524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;
1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction;(b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA. - View Dependent Claims (20, 21, 22, 23, 24)
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Specification
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Current AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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Original AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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InventorsKamtekar, Satwik, Miller, Erik
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Primary Examiner(s)Hutson, Richard G
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Application NumberUS15/188,672Publication NumberTime in Patent Office581 DaysField of SearchNoneUS Class CurrentCPC Class CodesC12N 9/1252 DNA-directed DNA polymerase...C12Q 1/686 Polymerase chain reaction [...C12Q 1/6869 Methods for sequencingC12Y 207/07007 DNA-directed DNA polymerase...
