Computer-directed assembly of a polynucleotide encoding a target polypeptide
First Claim
Patent Images
1. A method of synthesizing a target polynucleotide comprising:
- a) providing a target polynucleotide sequence;
b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang and a 3′
overhang;
c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating polynucleotide and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating polynucleotide;
d) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating polynucleotide which is not complementary to an overhang of the second polynucleotide;
e) contacting the initiating polynucleotide of b) with the second polynucleotide of c) and the third polynucleotide of d) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally;
f) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation; and
g) optionally repeating b) through f) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized.
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Abstract
The present invention outlines a novel approach to utilizing the results of genomic sequence information by computer-directed polynucleotide assembly based upon information available in databases such as the human genome database. Specifically, the present invention may be used to select, synthesize and assemble a novel, synthetic target polynucleotide sequence encoding a target polypeptide. The target polynucleotide may encode a target polypeptide that exhibits enhanced or altered biological activity as compared to a model polypeptide encoded by a natural (wild-type) or model polynucleotide sequence.
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Citations
52 Claims
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1. A method of synthesizing a target polynucleotide comprising:
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a) providing a target polynucleotide sequence;
b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang and a 3′
overhang;
c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating polynucleotide and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating polynucleotide;
d) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating polynucleotide which is not complementary to an overhang of the second polynucleotide;
e) contacting the initiating polynucleotide of b) with the second polynucleotide of c) and the third polynucleotide of d) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally;
f) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation; and
g) optionally repeating b) through f) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 38)
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14. A method of synthesizing a target polynucleotide comprising:
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a) providing a target polynucleotide sequence derived from a model sequence;
b) identifying at least one initiating polynucleotide sequence present in the target polynucleotide sequence of a), wherein the initiating polynucleotide comprises;
1) a first plus strand oligonucleotide;
2) a second plus strand oligonucleotide contiguous with the first plus strand oligonucleotide; and
3) a minus strand oligonucleotide comprising a first contiguous sequence that is at least partially complementary to the first plus strand oligonucleotide and second contiguous sequence which is at least partially complementary to the second plus strand oligonucleotide;
c) annealing the first plus strand oligonucleotide and the second plus strand oligonucleotide to the minus strand oligonucleotide of b) resulting in a partially double-stranded initiating polynucleotide comprised of a 5′
overhang and a 3′
overhang;
d) identifying a second polynucleotide sequence present in the target polynucleotide sequence of a), wherein the second polynucleotide sequence is contiguous with the initiating polynucleotide sequence and comprises;
1) a first plus strand oligonucleotide;
2) a second plus strand oligonucleotide contiguous with the first plus strand oligonucleotide; and
3) a minus strand oligonucleotide comprising a first contiguous sequence which is at least partially complementary to the first plus strand oligonucleotide and second contiguous sequence which is at least partially complementary to the second plus strand oligonucleotide;
e) annealing the first plus strand oligonucleotide and the second plus strand oligonucleotide to the minus strand oligonucleotide of d) resulting in a partially double-stranded second polynucleotide, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating polynucleotide;
f) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises;
1) a first plus strand oligonucleotide;
2) a second plus strand oligonucleotide contiguous with the first plus strand oligonucleotide; and
3) a minus strand oligonucleotide comprising a first contiguous sequence which is at least partially complementary to the first plus strand oligonucleotide and second contiguous sequence which is at least partially complementary to the second plus strand oligonucleotide;
g) annealing the first plus strand oligonucleotide and the second plus strand oligonucleotide to the minus strand oligonucleotide of f) resulting in a partially double-stranded second polynucleotide, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating polynucleotide and not complementary to an overhang of the second polynucleotide;
h) contacting the initiating polynucleotide of c) with the second polynucleotide of e) and the third polynucleotide of g) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally;
i) in the absence of primer extension, optionally contacting the mixture of h) with a ligase under conditions suitable for ligation; and
j) optionally repeating b) through i) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized.
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15. A method for synthesizing a target polynucleotide, comprising:
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a) providing a target polynucleotide sequence;
b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide;
c) contacting the initiating polynucleotide under conditions suitable for primer annealing with a first oligonucleotide having partial complementarity to the 3′
portion of the plus strand of the initiating polynucleotide, and a second oligonucleotide having partial complementarity to the 3′
portion of the minus strand of the initiating polynucleotide;
d) catalyzing under conditions suitable for primer extension;
1) polynucleotide synthesis from the 3′
-hydroxyl of the plus strand of the initiating polynucleotide;
2) polynucleotide synthesis from the 3′
-hydroxyl of the annealed first oligonucleotide;
3) polynucleotide synthesis from the 3′
-hydroxyl of the minus strand of the initiating polynucleotide; and
4) polynucleotide synthesis from the 3′
-hydroxyl of the annealed second oligonucleotide, wherein the initiating sequence is extended bi-directionally thereby forming a nascent extended initiating polynucleotide;
e) contacting the extended initiating polynucleotide of d) under conditions suitable for primer annealing with a third oligonucleotide having partial complementarity to the 3′
portion of the plus strand of the extended initiating polynucleotide, and a fourth oligonucleotide having partial complementarity to the 3′
portion of the minus strand of the extended initiating polynucleotide;
f) catalyzing under conditions suitable for primer extension;
1) polynucleotide synthesis from the 3′
-hydroxyl of the plus strand of the extended initiating polynucleotide;
2) polynucleotide synthesis from the 3′
-hydroxyl of the annealed third oligonucleotide;
3) polynucleotide synthesis from the 3′
-hydroxyl of the minus strand of the extended initiating polynucleotide; and
4) polynucleotide synthesis from the 3′
-hydroxyl of the annealed fourth oligonucleotide, wherein the extended initiating sequence is extended bi-directionally thereby forming a nascent extended initiating polynucleotide; and
g) optionally repeating. e) through f) as desired, resulting in formation of the target polynucleotide sequence. - View Dependent Claims (16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27)
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20. A method of synthesizing a target polynucleotide comprising:
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a) providing a target polynucleotide sequence;
b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of at least a 5′
overhang or a 3′
overhang;
c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating polynucleotide and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the second polynucleotide is complementary to the overhang of the initiating polynucleotide;
d) contacting the initiating polynucleotide of b) with the second polynucleotide of c) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended uni-directionally;
e) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation; and
f) optionally repeating b) through e) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized.
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28. A method for isolating a target polypeptide encoded by a target polynucleotide, comprising:
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a) providing a target polynucleotide sequence derived from a model sequence;
b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang and a 3′
overhang;
c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating sequence;
d) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′
overhang, a 3′
overhang, or a 5′
overhang and a 3′
overhang, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating sequence which is not complementary to an overhang of the second polynucleotide;
e) contacting the initiating polynucleotide of b) with the second polynucleotide of c) and the third polynucleotide of d) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally;
f) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation;
g) optionally repeating b) through f) to sequentially add double-stranded polynucleotides to the extended initiating sequence through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized;
h) incorporating the target polynucleotide of g) in an expression vector;
i) introducing the expression vector of h) into a suitable host cell;
j) culturing the cell of i) under conditions and for such time as to promote the expression of the target polypeptide encoded by the target polynucleotide; and
k) isolating the target polypeptide. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37)
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39. A method of synthesizing a target polynucleotide comprising:
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a) providing a target polynucleotide sequence derived from a model sequence;
b) chemically synthesizing a plurality of single-stranded oligonucleotides each of which is partially complementary to at least one oligonucleotide present in the plurality, wherein the sequence of the plurality of oligonucleotides is a contiguous sequence of the target polynucleotide;
c) contacting the partially complementary oligonucleotides of b) under conditions and for such time suitable for annealing, the contacting resulting in a plurality of partially double-stranded polynucleotides, wherein each double-stranded polynucleotide is comprised of a 5′
overhang and a 3′
overhang;
d) identifying at least one initiating polynucleotide derived from the model sequence, wherein the initiating polynucleotide is present in the plurality of double-stranded polynucleotides set forth in c);
e) in the absence of primer extension, subjecting a mixture comprising the initiating polynucleotide and
1) a double-stranded polynucleotide that will anneal to the 5′
portion of said initiating and sequence;
2) a double-stranded polynucleotide that will anneal to the 3′
portion of the initiating polynucleotide; and
3) a DNA ligase under conditions suitable for annealing and ligation, wherein the initiating polynucleotide is extended bi-directionally;
f) sequentially annealing double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing, whereby the target polynucleotide is produced. - View Dependent Claims (40)
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41. A computer program, stored on a computer-readable medium, for generating a target polynucleotide sequence, the computer program comprising instructions for causing a computer system to:
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a) identify an initiating polynucleotide sequence contained in the target polynucleotide sequence;
b) parse the target polynucleotide sequence into multiply distinct, partially complementary, oligonucleotides;
c) control assembly of the target polynucleotide sequence by controlling the bi-directional extension of the initiating polynucleotide sequence by the sequential addition of partially complementary oligonucleotides resulting in a contiguous double-stranded polynucleotide. - View Dependent Claims (42, 43, 44)
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45. A computer-assisted method for synthesizing a target polynucleotide encoding a target polypeptide derived from a model sequence using a programmed computer including a processor, an input device, and an output device, comprising:
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a) inputting into the programmed computer, through the input device, data including at least a portion of the target polynucleotide sequence encoding a target polypeptide;
b) determining, using the processor, the sequence of at least one initiating polynucleotide present in the target polynucleotide sequence c) selecting, using the processor, a model for synthesizing the target polynucleotide sequence based on the position of the initiating sequence in the target polynucleotide sequence using overall sequence parameters necessary for expression of the target polypeptide in a biological system; and
d) outputting, to the output device, the results of the at least one determination. - View Dependent Claims (46)
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47. A method for automated synthesis of a target polynucleotide sequence, comprising:
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a) providing a user with an opportunity to communicate a desired target polynucleotide sequence;
b) allowing the user to transmit the desired target polynucleotide sequence to a server;
c) providing the user with a unique designation;
d) obtaining the transmitted target polynucleotide sequence provided by the user. - View Dependent Claims (48, 49)
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50. A method for automated synthesis of a polynucleotide, comprising:
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a) providing a user with a mechanism for communicating a model polynucleotide sequence;
b) optionally providing the user with an opportunity to communicate at least one desired modification to the model sequence if desired;
c) allowing the user to transmit the model sequence and desired modification to a server;
d) providing user with a unique designation;
e) obtaining the transmitted model sequence and desired modification provided by the user;
f) inputting into a programmed computer, through an input device, data including at least a portion of the model polynucleotide sequence;
g) determining, using the processor, the sequence of the model polynucleotide sequence containing the desired modification;
h) further determining, using the processor, at least one initiating polynucleotide sequence present in the model polynucleotide sequence i) selecting, using the processor, a model for synthesizing the modified model polynucleotide sequence based on the position of the initiating sequence in the model polynucleotide sequence; and
j) outputting, to-the output device, the results of the at least one determination.
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51. An isolated polynucleotide composition comprising:
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a) an initiating polynucleotide comprising a plus strand and a minus strand, wherein the plus or minus strand is modified to incorporate a moiety that binds to a solid support;
b) a first primer suitable for primer extension having partial complementarity to the 3′
portion of the plus strand of the initiating polynucleotidec) a second primer suitable for primer extension having partial complementarity to the 3′
portion of the minus strand of the initiating polynucleotide; and
d) a solid support matrix, wherein each of the first and second primers consists of about 25 to 1000 nucleotides.
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52. An isolated polynucleotide composition comprising:
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a) an initiating polynucleotide comprising a plus strand and a minus strand, wherein the plus or minus strand is modified to incorporate a moiety that binds to a solid support;
b) a first primer suitable for primer extension having partial complementarity to the 3′
portion of the plus strand of the initiating polynucleotidec) a second primer suitable for primer extension having partial complementarity to the 3′
portion of the minus strand of the initiating polynucleotide; and
d) a solid support matrix, wherein each of the first and second primers consists of about 25 to 1000 nucleotides.
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Specification