Methods for purifying annealed double-stranded oligonucleotides lacking base pair mismatches or nucleotide gaps
First Claim
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1. A method for purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps comprising the following steps:
- (a) providing a plurality of polypeptides that specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps within a double stranded polynucleotide comprising a Vsr (very short patch repair) bacterial DNA mismatch repair enzyme;
(b) providing a sample comprising a plurality of double-stranded polynucleotides;
(c) contacting the double-stranded polynucleotides of step (b) with the polypeptides of step (a) under conditions wherein a polypeptide of step (a) can specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps in a double-stranded polynucleotide of step (b); and
(d) separating the double-stranded polynucleotides lacking a specifically bound polypeptide of step (a) from the double-stranded polynucleotides to which a polypeptide of step (a) has specifically bound, thereby purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps.
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Abstract
The invention provides methods for identifying and purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or nucleotide gaps.
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Citations
34 Claims
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1. A method for purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps comprising the following steps:
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(a) providing a plurality of polypeptides that specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps within a double stranded polynucleotide comprising a Vsr (very short patch repair) bacterial DNA mismatch repair enzyme;
(b) providing a sample comprising a plurality of double-stranded polynucleotides;
(c) contacting the double-stranded polynucleotides of step (b) with the polypeptides of step (a) under conditions wherein a polypeptide of step (a) can specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps in a double-stranded polynucleotide of step (b); and
(d) separating the double-stranded polynucleotides lacking a specifically bound polypeptide of step (a) from the double-stranded polynucleotides to which a polypeptide of step (a) has specifically bound, thereby purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps comprising the following steps:
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(a) providing a plurality of polypeptides that specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps within a double-stranded polynucleotide;
(b) providing a sample comprising a plurality of double-stranded polynucleotides encoding a fusion protein comprising a polypeptide of interest upstream of an intein, wherein the intein encodes a polypeptide;
(c) contacting the double-stranded polynucleotides of step (b) with the polypeptides of step (a) under conditions wherein a polypeptide of step (a) can specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps in a double-stranded polynucleotide of step (b); and
(d) separating the double-stranded polynucleotides lacking a specifically bound polypeptide of step (a) from the double-stranded polynucleotides to which a polypeptide of step (a) has specifically bound, thereby purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps. - View Dependent Claims (22, 23, 24, 25, 26, 27)
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28. A method for purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps comprising:
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(a) providing a plurality of polypeptides that specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps within a double stranded polynucleotide;
(b) providing a sample comprising a plurality of double-stranded polynucleotides encoding a fusion protein comprising a polypeptide of interest and an intein, wherein the intein encodes a polypeptide;
(c) contacting the double-stranded polynucleotides of (b) with the polypeptides of (a) under conditions wherein a polypeptide of (a) can specifically bind to a base pair mismatch, an insertion/deletion loop and/or a nucleotide gap or gaps in a double stranded polynucleotide of (b); and
(d) separating the double-stranded polynucleotides lacking a specifically bound polypeptide of (a) from the double-stranded polynucleotides to which a polypeptide of (a) has specifically bound, thereby purifying double-stranded polynucleotides lacking base pair mismatches, insertion/deletion loops and/or a nucleotide gap or gaps. - View Dependent Claims (29, 30, 31, 32, 33, 34)
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Specification