Devices comprising a polynucleotide to extend single stranded target molecules

1. A device for extension of a single stranded target molecule, wherein the device is a polynucleotide comprising Structure 1:

wherein:

P is a primer sequence through which at least the 3′ end of the target molecule hybridizes to the device;

Y is product sequence;

R is replication blocking group;

S is a stem sequence;

L is a loop region;

S′ is a sequence which hybridizes to S;

Y′ is a sequence which hybridizes to Y; and

wherein each of S and S′ is optionally present in the device, wherein the presence of S′ is dependent on the presence of S in the device,

wherein Y, R, S, L, S′, and Y′ form a hairpin structure,

wherein Y comprises or consists of a sequence P Y which is identical to priming sequence P.

2. A device for extension of a single stranded target molecule, the device comprising a first polynucleotide of Structure 2:

and a second polynucleotide of Structure 3:

wherein:

P is a primer sequence through which at least the 3′ end of the target molecule hybridizes to the device;

Y is product sequence;

R is replication blocking group;

S is a stem sequence;

Y′ is a sequence which hybridizes to Y;

S′ is a sequence which hybridizes to S;

wherein Y comprises or consists of a sequence P Y which is identical to priming sequence P, optionally, wherein sequence S and sequence S′ form a duplex structure which is stable at a temperature between about 20° C. and about 95° C. or between about 60° C. and about 75° C.

3. The device of claim 1 or 2 :

(1) wherein P is about 6 bases to about 30 bases in length,

(2) wherein Y further comprises a tag sequence T which is different from the sequence P Y and the tag sequence T is located 3′ to sequence P Y and 5′ to primer sequence P, optionally, wherein sequence T is about 15 bases to about 50 bases in length;

(3) comprising a 3′ blocking group F which blocks extension of a sequence of the device, wherein F is located 3′ to P, optionally, wherein F is an amino group, a phosphate, or a dideoxynucleotide;

(4) comprising one or more modified internucleotide linkages which are not cleavable by an enzyme, optionally, wherein the one or more modified internucleotide linkages are located 5′ to product sequence Y; and/or

(5) comprising a sequence X located 5′ to Y′ and comprising a nucleotide sequence of about 1 base to about 25 bases in length, wherein X (i) does not hybridize to P, (ii) hybridizes to at least a portion of P, or (iii) hybridizes to at least a portion of P and to at least a portion of Y, optionally, wherein X hybridizes to only a portion of P or hybridizes to only a portion of P and to only a portion of Y or X does not hybridize to P and comprises a nucleotide sequence of about 1 to about 10 bases in length.

4. The device of claim 1 or claim 2 , wherein R is (i) an abasic site, (ii) a modified base, (iii) a base which is absent from product sequence Y or is present in a reaction mixture at a limiting concentration, or (iv) a spacer.

5. The device of claim 4 ,

(1) wherein the modified base comprises a chemical moiety which sterically hinders a polymerase activity to or beyond R;

(2) wherein the modified base is a base which is cross-linked to another base of the oligonucleotide;

(3) wherein the modified base is cross-linked to a base of S′;

(4) wherein the abasic site does not specifically bind to a base of sequence S′ or sequence Y′; and/or

(5) wherein the spacer is a hexamethylene glycol spacer, a hexylene glycol spacer, or a 2-deoxyribose spacer.

6. The device of claim 1 or claim 2 , wherein the sequence of S comprises a GC content between about 0% to about 100%, between about 70% and about 100% or between about 80% and about 100%.

7. A composition comprising at least two devices according to claim 1 or claim 2 , optionally, (i) wherein at least two devices in the composition comprise a first device comprising a product sequence Y 1 and a second device comprising a product sequence Y 2 , wherein Y 1 is different from Y 2 , or (ii) wherein the composition comprises a plurality of devices of claim 1 or claim 2 , wherein the plurality comprises at least three subsets of devices, wherein each device of a subset comprises a product sequence Y which is (i) the same as the sequence Y of another device of the same subset and (ii) different from the sequence Y of a device of another subset of the plurality.

8. A composition comprising at least two devices, wherein each device (i) is a polynucleotide comprising Structure 1:

wherein:

P is a primer sequence through which at least the 3′ end of the target molecule hybridizes to the device;

Y is product sequence;

R is replication blocking group;

S is a stem sequence;

L is a loop region;

S′ is a sequence which hybridizes to S;

Y′ is a sequence which hybridizes to Y; and

wherein each of S and S′ is optionally present in the device, wherein the presence of S′ is dependent on the presence of S in the device,

wherein Y, R, S, L, S′, and Y′ form a hairpin structure,

or (ii) comprises a first polynucleotide of Structure 2:

and a second polynucleotide of Structure 3:

wherein:

P is a primer sequence through which at least the 3′ end of the target molecule hybridizes to the device;

Y is product sequence;

R is replication blocking group;

S is a stem sequence;

Y′ is a sequence which hybridizes to Y;

S′ is a sequence which hybridizes to S;

wherein each device of the composition comprises a product sequence Y consisting of an additional sequence A which is not identical to priming sequence P,

wherein the composition comprises a first device comprising a primer sequence P 1 and a product sequence Y 1 and a second device comprising a primer sequence P 2 and a product sequence Y 2 , wherein P 1 is different from P 2 and Y 1 is different from Y 2 , wherein the complement of Y 1 is complementary to primer sequence P 2 ;

optionally, wherein the composition comprises a plurality of devices, wherein the plurality comprises at least three subsets of devices, wherein each device of each subset comprises a product sequence Y which is (i) the same as the sequence Y of another device of the same subset and (ii) different from the sequence Y of a device of another subset of the plurality.

9. A kit comprising a device of claim 1 or claim 2 , or a composition of claim 8 , and instructions for using the device or composition in a reaction which extends a single stranded primer.

10. The composition of claim 8 , further comprising a polymerase and free nucleotides, wherein the polymerase is selected from the group consisting of a strand displacement polymerase, a strand displacement polymerase which operates under substantially isothermal conditions, a DNA polymerase, an RNA polymerase, a 5′ exonuclease-minus polymerase, a polymerase that dissociates from the device when in contact with replication blocking group R, a polymerase that dissociates from the device under substantially isothermal conditions, and at least two polymerases, wherein at least one polymerase is a proofreading polymerase, optionally, wherein the proofreading polymerase removes bases from the single stranded target molecule that are added via one or more non-template addition reactions.

11. A method of extending a single stranded target molecule, comprising contacting the single stranded target molecule with an extension reaction mixture comprising (i) a device of claim 1 or claim 2 or a composition of claim 8 , (ii) a polymerase, and (iii) free nucleotides, whereupon an extension reaction product is generated, wherein the extension reaction product comprises the single stranded target molecule with a 3′ sequence complementary to product sequence Y of the device.

12. A method of extending a single stranded target molecule, comprising contacting the single stranded target molecule with an extension reaction mixture comprising (i) a composition of claim 8 , (ii) a polymerase, and (iii) free nucleotides, whereupon an extension reaction product is generated, wherein the extension reaction product comprises the single stranded target molecule with a 3′ sequence complementary to product sequence Y of the device,

wherein the extension reaction product generated from a first extension reaction using the first device is further extended by a second extension reaction using the second device, wherein the extension reaction product includes a 3′ terminal sequence 5′(cP1-cY1-cY2)3′, wherein cP1 is a sequence which is complementary to P1, cY1 is a sequence which is complementary to Y1 and cY2 is a sequence which is complementary to Y2, or wherein the extension reaction mixture comprises a composition of claim 8 , wherein the extension reaction product generated from a first extension reaction using a device of a first subset is further extended by a second extension reaction using a device of a second subset, wherein the extension reaction product generated from a second extension reaction using a device from a third subset, optionally, wherein (i) the extension reaction mixture comprises n subsets of devices, (ii) the method comprises n extension reactions in which a sequence complementary to additional sequence A of the device used in the extension reaction is consecutively added to the 3′ end of the target molecule, and (iii) the extension reaction product generated from the n th extension reaction comprises n different added sequences, each added sequence is complementary to additional sequence A of a device of each subset.

13. A method of extending a single stranded target molecule, comprising contacting the single stranded target molecule with an extension reaction mixture comprising (i) a device of claim 1 or claim 2 , (ii) a polymerase, and (iii) free nucleotides, whereupon an extension reaction product is generated, wherein the extension reaction product comprises the single stranded target molecule with a 3′ sequence complementary to product sequence Y of the device,

wherein product sequence Y of the device consists of a sequence P Y which is identical to primer sequence P of the device, wherein at least a portion of the target molecule sequence is complementary to the primer sequence P, and wherein the extension reaction product has a 3′ terminal sequence that is complementary to the primer sequence P, and optionally further comprising n extension reactions and the extension reaction product generated from the n th extension reaction comprises (n+1) cP sequences, wherein cP is a sequence complementary to the primer sequence P.

14. A method of extending a single stranded target molecule, comprising contacting the single stranded target molecule with an extension reaction mixture comprising (i) a device of claim 1 or claim 2 :

(1) wherein P is about 6 bases to about 30 bases in length,

(2) wherein Y further comprises a tag sequence T which is different from the sequence PY and the tag sequence T is located 3′ to sequence PY and 5′ to primer sequence P, optionally, wherein sequence T is about 15 bases to about 50 bases in length;

(3) comprising a 3′ blocking group F which blocks extension of a sequence of the device, wherein F is located 3′ to P, optionally, wherein F is an amino group, a phosphate, or a dideoxynucleotide;

(4) comprising one or more modified internucleotide linkages which are not cleavable by an enzyme, optionally, wherein the one or more modified internucleotide linkages are located 5′ to product sequence Y; and/or

(5) comprising a sequence X located 5′ to Y′ and comprising a nucleotide sequence of about 1 base to about 25 bases in length, wherein X (i) does not hybridize to P, (ii) hybridizes to at least a portion of P, or (iii) hybridizes to at least a portion of P and to at least a portion of Y, optionally, wherein X hybridizes to only a portion of P or hybridizes to only a portion of P and to only a portion of Y or X does not hybridize to P and comprises a nucleotide sequence of about 1 to about 10 bases in length;

(ii) a polymerase, and (iii) free nucleotides, whereupon an extension reaction product is generated, wherein the extension reaction product comprises the single stranded target molecule with a 3′ sequence complementary to product sequence Y of the device,

wherein product sequence Y of the device comprises sequence P Y which is identical to primer sequence P of the device and further comprises a tag sequence T which is different from the sequence of sequence P Y and sequence T is located 3′ to P Y , and optionally further comprising n extension reactions and the extension reaction product generated from the n th extension reaction comprises cP followed by n number of (cT-cP) sequences, wherein cP is a sequence complementary to the primer sequence P and cT-cP is a sequence complementary to the tag sequence T followed by a sequence complementary to the primer P, or wherein the extension reaction mixture comprises a plurality of devices of claim 1 or claim 2 , wherein the plurality comprises at least three subsets of devices, wherein each device of a subset comprises a sequence Y sequence which is (i) the same as sequence Y of another device of the same subset and (ii) different from sequence Y of a device of another subset of the plurality, wherein the extension product has a 3′ terminus which comprises a sequence which is complementary to each of sequence Y of the plurality.

15. The method of claim 11 , wherein the method is carried out under substantially isothermal conditions, optionally within a range of about 20° C. to about 95° C.

16. The method of claim 11 , wherein the method comprises one or more steps carried out at a first temperature and one or more steps carried out at a second temperature, wherein the second temperature is at least or about 25° C. greater than the first temperature, optionally wherein the first temperature is within a range of about 60° C. to about 75° C. and the second temperature is within a range of about 85° C. to about 100° C., and/or comprising a dissociation step in which the polymerase dissociates from the device at the second temperature.