Methods for polynucleotide synthesis and articles for polynucleotide hybridization
First Claim
1. A method for synthesizing a polynucleotide comprising the following steps:
- a) obtaining a double-stranded polynucleotide wherein one or more nucleotides at one end of one strand (α
) extend as a single-stranded structure beyond the end of the other (β
) strand to form a sticky end;
b) obtaining a third polynucleotide strand (γ
) which has a nucleotide sequence at one end which can hybridize to the sticky end of the α
polynucleotide, wherein the bases of a number p of nucleotides in the γ
strand adjacent to the terminal nucleotide sequence complementary to the sticky end of the α
strand are universal bases, where p is a number of new nucleotides to be added to the end of the α
strand,hybridizing the γ
strand to said sticky end, andligating the hybridized end of the γ
strand to the end of the β
strand;
c) obtaining a fourth polynucleotide strand (ε
) which is complementary to the single-stranded portion of the ligated γ
strand, wherein the ε
strand has p nucleotides at its end which are to be added to the α
strand, which nucleotides pair with the p universal bases of the γ
strand andhybridizing the ε
strand to the γ
strand, andligating the end of the hybridized ε
strand to the adjacent end of the α
strand;
d) cutting the resulting double-stranded DNA complexes with a restriction enzyme so that the end of the α
strand has p bases more than it had in step a, and so that one or more nucleotides of the lengthened end of the α
strand extend beyond the end of the β
strand to form a sticky end; and
e) repeating steps b-d until the newly synthesized polynucleotide has the desired nucleotide sequence.
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Abstract
This invention relates to methods for making polynucleotides of any desired nucleotide sequence by repeating a series of reactions involving assembly of overlapping oligomers, ligation by DNA ligase, and cleavage with a restriction enzyme that cuts so as to add one or more additional nucleotides to a growing polynucleotide. This invention also relates to combining such a method for polynucleotide synthesis with a step employing localized melting of hybridized DNA oligomers, to synthesize an array of polynucleotides of different, defined nucleotide sequence on a substrate, and to the articles for polynucleotide hybridization so made. This invention further includes methods in which a substrate bearing an array of polynucleotides made according to the invention is used to determine the nucleotide sequence of a nucleic acid, or to detect or isolate a nucleic acid having a selected nucleotide sequence, and a device which analyzes a substrate-bound array of polynucleotides made according to the invention to determine the nucleotide sequence of a nucleic acid, or to detect or isolate a nucleic acid having a selected nucleotide sequence.
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Citations
28 Claims
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1. A method for synthesizing a polynucleotide comprising the following steps:
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a) obtaining a double-stranded polynucleotide wherein one or more nucleotides at one end of one strand (α
) extend as a single-stranded structure beyond the end of the other (β
) strand to form a sticky end;b) obtaining a third polynucleotide strand (γ
) which has a nucleotide sequence at one end which can hybridize to the sticky end of the α
polynucleotide, wherein the bases of a number p of nucleotides in the γ
strand adjacent to the terminal nucleotide sequence complementary to the sticky end of the α
strand are universal bases, where p is a number of new nucleotides to be added to the end of the α
strand,hybridizing the γ
strand to said sticky end, andligating the hybridized end of the γ
strand to the end of the β
strand;c) obtaining a fourth polynucleotide strand (ε
) which is complementary to the single-stranded portion of the ligated γ
strand, wherein the ε
strand has p nucleotides at its end which are to be added to the α
strand, which nucleotides pair with the p universal bases of the γ
strand andhybridizing the ε
strand to the γ
strand, andligating the end of the hybridized ε
strand to the adjacent end of the α
strand;d) cutting the resulting double-stranded DNA complexes with a restriction enzyme so that the end of the α
strand has p bases more than it had in step a, and so that one or more nucleotides of the lengthened end of the α
strand extend beyond the end of the β
strand to form a sticky end; ande) repeating steps b-d until the newly synthesized polynucleotide has the desired nucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for making an article for nucleic acid hybridization comprising a substrate-bound array of hybridizing polynucleotides that have different, selected nucleotide sequences, the method comprising the steps of:
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a) covering part or all of a substrate with double-stranded polynucleotides comprising complementary α and
β
polynucleotide strands hybridized to each other, wherein the 3'"'"' ends of the α
strands are anchored to the substrate and the 5'"'"' ends of the α
strands are unanchored and phosphorylated, wherein the 3'"'"' end of each β
strand terminates in a hydroxyl group, and wherein one or more nucleotides at the 5'"'"' end of each α
strand extend as a single-stranded structure beyond the 3'"'"' end of the β
strand to form a sticky end;b) hybridizing to said sticky ends a third polynucleotide strand (γ
) which has a nucleotide sequence at its 5'"'"' end which is complementary to the sticky end of the α
strand, wherein the bases of a number p of nucleotides in the γ
strand adjacent to said terminal nucleotide sequence complementary to the sticky end of the α
strand are universal bases, where p is a number of new nucleotides to be added to the end of each α
strand, andligating the hybridized 5'"'"' ends of the γ
strands to the adjacent 3'"'"' ends of the β
strands;c) hybridizing to said γ
strands a fourth polynucleotide strand (ε
) which is complementary to the single-stranded portion of each ligated γ
strand, wherein each ε
strand has p nucleotides at its 3'"'"' end which are to be added to the 5'"'"' end of an α
strand, which nucleotides pair with the p universal bases on each γ
strand;d) locally heating those pixels on the substrate where a different selected sequence of p nucleotides is to be added, to remove the undesired ε
strands from the ligated γ
strands in those pixels;e) hybridizing to the single stranded γ
strands in the heated pixels ε
strands which have at their 3'"'"' ends a selected sequence of p nucleotides which is different from the sequence of p nucleotides added previously;f) repeating steps d and e until ε
oligomers with desired sequences have been hybridized to all pixels where nucleotides are to be added;g) ligating the 3'"'"' ends of the hybridized ε
strands to the adjacent 5'"'"' ends of the α
strands;h) cutting the resulting double-stranded DNA complexes with a restriction enzyme so that the end of each α
strand has p bases more than it had in step a, and so that one or more nucleotides of the lengthened 5'"'"' end of the α
strand extend beyond the 3'"'"' end of the β
strand to form a new sticky end; ande) repeating steps b-h until the polynucleotides in each pixel on the substrate have the desired nucleotide sequence. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification