Strand displacing amplification enzymes

1 . A polymerase comprising an amino acid sequence that is at least 80% identical to a contiguous 500 amino acid sequence within SEQ ID NO: 1; comprising

a first substitution at amino acid position 588 or an amino acid position corresponding to position 588; wherein the first substitution is leucine, isoleucine, valine, alanine, or glycine; and

a second substitution:

at amino acid position 7 or an amino acid position corresponding to position 7, wherein the second substitution is histidine, lysine, or arginine;

at amino acid position 97 or an amino acid position corresponding to position 97, wherein the second substitution is cysteine, histidine, lysine, serine, threonine, or methionine; or

at amino acid position 742 or an amino acid position corresponding to position 742, wherein the second substitution is leucine, isoleucine, alanine, or glycine.

2 . The polymerase of claim 1 , wherein the second substitution is a substitution at amino acid position 7 or an amino acid position corresponding to position 7, wherein the substitution is histidine, lysine, or arginine.

3 . The polymerase of claim 2 , further comprising a substitution at amino acid position 742 or an amino acid position corresponding to position 742, wherein the substitution is leucine, isoleucine, alanine, or glycine.

4 . The polymerase of claim 1 , wherein the second substitution is a substitution at amino acid position 97 or an amino acid position corresponding to position 97, wherein the substitution is cysteine, histidine, lysine, serine, threonine, or methionine.

5 . The polymerase of claim 4 , further comprising a substitution at amino acid position 742 or an amino acid position corresponding to position 742, wherein the substitution is leucine, isoleucine, alanine, or glycine.

6 . The polymerase of claim 1 , wherein the second substitution is a substitution at amino acid position 742 or an amino acid position corresponding to position 742, wherein the substitution is leucine, isoleucine, alanine, or glycine.

7 . The polymerase of claim 1 , comprising a substitution at amino acid position 13 or an amino acid position corresponding to position 13, wherein the substitution is arginine, isoleucine, methionine, or histidine.

8 . The polymerase of claim 1 , wherein the second substitution is a substitution at amino acid position 97 or an amino acid position corresponding to position 97, wherein the substitution is cysteine or histidine.

9 . The polymerase of claim 1 , wherein the first substitution is leucine, isoleucine, valine, or alanine.

10 . The polymerase of claim 1 , wherein the first substitution is leucine or valine.

11 . The polymerase of claim 1 , comprising a substitution at amino acid position 579 or an amino acid position corresponding to position 579, wherein the substitution is alanine or glycine.

12 . The polymerase of claim 1 , comprising a substitution at amino acid position 726 or an amino acid position corresponding to amino acid position 726, wherein the substitution is aspartic acid, glutamic acid, asparagine, or glutamine.

13 . The polymerase of claim 1 , comprising a substitution at amino acid position 13 or an amino acid position corresponding to position 13, wherein the substitution is arginine, leucine, isoleucine, or histidine.

14 . The polymerase of claim 1 , comprising:

a substitution at amino acid position 13 or an amino acid position corresponding to position 13, wherein the substitution is arginine, leucine, isoleucine, or histidine; and

a substitution at amino acid position 579 or an amino acid position corresponding to position 579, wherein the substitution is alanine or glycine.

15 . The polymerase of claim 1 , comprising:

R97H, F588L, G635D, and V742A;

K13R, R97C, E579G, and F588L;

R97C, E563G, E579G, F588L, and V742A;

R97H, F588L, and G635D;

R97C, F588L, and V742A;

Y7H, K13I, E579G, F588L, and V742A;

Y7H, K13R, R97H, and V742A;

Y7H, K13R, R97C, D141A, E579G, F588L, and V742A;

R97C, E579G, F588L, and V742A;

Y7H, R97C, E579G, and F588L;

Y7H, R97C, and V742A; or

Y7H, K13R, E563G, F588L, and V742A.

16 . The polymerase of claim 1 , comprising:

E29D, Y30F, R97H, I160V, K229E, A511V, 1548V, F588L, G635D, and V742A;

K13R, R32H, F75L, R97C, K201I, Y209H, 1256V, Y291H, E383D, G400D, R526C, E579G, F588L, E638G, and A730V;

F75L, R97C, I142T, V278I, A281T, A292E, M329L, P372S, H440N, E563G, E579G, F588L, D729Y, and V742A;

I65V, R97H, F283S, V308M, K465E, F588L, T591A, I604M, G635D, and K727T;

E25K, R97C, A168T, R255H, F326Y, K478R, R526H, E556K, K558N, P573S, E581N, F588L, E600K, E601K, R686H, R724H, V742A, K752E, and K762N;

Y7H, K13I, F75L, R97L, I160V, V170I, A316P, K469E, A491V, E579G, F588L, V637D, H726D, and V742A;

Y7H, K13R, A40V, R97H, F116L, A117S, K154E, A281T, I415V, K477I, K552N, N569S, E577K, A585G, F588I, E655D, and V742A;

Y7H, K13R, I38F, A40V, R97C, D141A, P286L, G350S, R467C, and K469T, E579G, F588L, F721Y, H726D, V742A, L756C, R757A, W758G, Q759R, T761P, K762N, Q763R, V764L, and G765V;

V63A, F75L, R97C, D98G, R188H, F214I, G245S, D246E, T319I, G350S, E579G, F588L, R690H, H726D, V742A, and K760N;

Y7H, I18V, R97C, A168S, F214S, G284S, P286Q, A292V, G304D, K391I, E431D, K477I, Y567H, E579G, F588L, and H726D;

Y7H, 18V, N23D, V66A, F75L, R97C, P217Q, A298S, A316P, K465E, R526H, E577K, E601D, T606I, E655D, R706C, V742A, and W769S; or

Y7H, K13R, L76P, K192E, K289T, R364C, L397M, Q484H, E563G, F588L, V637D, R706H, V742A, and L766P.

17 . The polymerase of claim 1 , further comprising an alanine at amino acid position 141 or an amino acid position corresponding to position 141; and an alanine at amino acid position 143 or an amino acid position corresponding to position 143.

18 . A method of incorporating a nucleotide into a nucleic acid sequence comprising combining in a reaction vessel: (i) a nucleic acid template, (ii) a nucleotide solution, and (iii) a polymerase, wherein the polymerase is a polymerase of claim 1 .

19 . A method of amplifying a nucleic acid sequence comprising:

a. hybridizing a nucleic acid template with a primer to form a primer-template hybridization complex;

b. contacting the primer-template hybridization complex with a DNA polymerase and a plurality of nucleotides, wherein the DNA polymerase is the polymerase of claim 1 ;

c. incorporating one or more nucleotides into the primer-template hybridization complex with the DNA polymerase to generate amplification products, thereby amplifying a nucleic acid sequence.

20 . A kit comprising the polymerase of claim 1 .

21 . The polymerase of claim 1 , comprising an amino acid sequence that is 85% identical to SEQ ID NO: 1.