Nucleic acid assembly optimization using clamped mismatch binding proteins
First Claim
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1. A method of preparing a nucleic acid having a predetermined sequence, the method comprising:
- assembling a sample of double-stranded target nucleic acids from a plurality of different starting nucleic acids in a multiplex assembly reaction;
denaturing and reannealing the sample of double stranded target nucleic acids, thereby forming a heterogeneous pool of reannealed double-stranded heteroduplex and homoduplex nucleic acids if the sample contains a mixture of error-free and error-containing target nucleic acid molecules;
contacting the reannealed double-stranded nucleic acids with a MutS or MutS homolog in the presence of ADP for a time and under conditions that allow the MutS or MutS homolog to bind to heteroduplex nucleic acids, increasing ATP concentration, after the reannealed double-stranded nucleic acids are circularized, to an ATP concentration that promotes the formation of a clamped form of the MutS or MutS homolog, and enriching for double-stranded homoduplex nucleic acids by selectively removing circularized double-stranded heteroduplex nucleic acids bound to the MutS or MutS homolog.
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Abstract
Certain aspects of the present invention provide methods for increasing the fidelity of multiplex nucleic acid assembly reactions using one or more mismatch binding proteins. Aspects of the invention also provide kits, compositions, devices, and systems for enriching nucleic acid samples for nucleic acids containing correct sequences using clamped forms of one or more mismatch binding proteins.
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Citations
25 Claims
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1. A method of preparing a nucleic acid having a predetermined sequence, the method comprising:
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assembling a sample of double-stranded target nucleic acids from a plurality of different starting nucleic acids in a multiplex assembly reaction;
denaturing and reannealing the sample of double stranded target nucleic acids, thereby forming a heterogeneous pool of reannealed double-stranded heteroduplex and homoduplex nucleic acids if the sample contains a mixture of error-free and error-containing target nucleic acid molecules;
contacting the reannealed double-stranded nucleic acids with a MutS or MutS homolog in the presence of ADP for a time and under conditions that allow the MutS or MutS homolog to bind to heteroduplex nucleic acids, increasing ATP concentration, after the reannealed double-stranded nucleic acids are circularized, to an ATP concentration that promotes the formation of a clamped form of the MutS or MutS homolog, and enriching for double-stranded homoduplex nucleic acids by selectively removing circularized double-stranded heteroduplex nucleic acids bound to the MutS or MutS homolog. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25)
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19-20. -20. (canceled)
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