Production of nucleic acid
A method for the production of nucleic acid encoding a target protein. The method comprises (a) providing an array of RNA or DNA molecules including one or more encoding the target protein; (b) generating a target protein from the array to form RNA-protein or DNA-protein complexes in which the RNA or DNA molecule is non-covalently or covalently bound to the complex; (c) separating the complexes into compartments wherein most or all of the compartments contain no more than one complex; (d) subjecting the complexes to reaction conditions which allow target protein activity; and (e) selecting nucleic acid encoding the target protein on the basis of the activity associated therewith, wherein when the complex is a DNA-protein complex in which the DNA is non-covalently bound, step b) is performed in the absence of separate compartments for each complex.
1. A reverse transcriptase comprising a Moloney Murine Leukemia Virus reverse transcriptase comprising the amino acid sequence as set forth in SEQ ID NO: 25, wherein the amino acid sequence comprises a mutation at at least position T330.
2. The reverse transcriptase according to claim 1 , wherein the mutation at T330 is T330P, N, L, D, V, or S.
3. The reverse transcriptase according to claim 2 , wherein the mutation is T330P.
4. The reverse transcriptase of claim 1 further comprising at least one mutation at an amino acid position chosen from:
E5,
M39,
I49,
M66,
Q91,
P130,
L139,
I179,
D200,
Q221,
Q237,
T287,
A307,
L333,
Y344,
Q374,
W388,
R390,
Q430,
D449,
N479,
A502,
H594,
L603,
E607,
A644,
N649,
D653,
K658,
L671,
and E673,
wherein when the further mutation is at position D653 the mutation is not D653N, and
wherein when the further mutation is at position H594 the mutation is not H594A.
5. The reverse transcriptase of claim 1 further comprising at least one mutation at an amino acid position chosen from L139, D200, D449, N479, or H594.
6. The reverse transcriptase of claim 5 , wherein the amino acid sequence comprises a mutation at L139.
7. The reverse transcriptase of claim 5 , wherein the amino acid sequence comprises a mutation at D200.
8. The reverse transcriptase of claim 5 , wherein the amino acid sequence comprises a mutation at D449.
9. The reverse transcriptase of claim 5 , wherein the amino acid sequence comprises a mutation at N479.
10. The reverse transcriptase of claim 5 , wherein the amino acid sequence comprises a mutation at H594.
11. The reverse transcriptase according to claim 2 , wherein the amino acid sequence comprises one of more of the following mutations:
E5K,
M39V or L,
I49V or T,
M66L,
Q91R or L,
P130S,
L139P, I179T or V,
D200N, A, or
G,
Q221R,
Q237R,
T287A,
A307V,
L333Q,
Y344H,
Q374R,
W388R,
R390W,
Q430R,
D449G or A,
N479D,
A502V,
H594R or Q,
L603W or M,
E607K, G, or
A,
A644V or T,
N649S,
D653G, A, H, or V,
K658R or Q,
L671P,
and E673G or
K.
12. The reverse transcriptase of claim 1 , wherein the reverse transcriptase has increased thermostability.
13. The reverse transcriptase of claim 1 , wherein the reverse transcriptase has an optimum activity at a temperature above 42° C.
14. The reverse transcriptase of claim 1 , wherein the reverse transcriptase has an optimum activity at a temperature of at least 50° C.
15. The reverse transcriptase of claim 1 , wherein the reverse transcriptase has a higher activity at 50° C. as compared with the corresponding wild type reverse transcriptase.
16. The reverse transcriptase according to claim 1 , wherein the reverse transcriptase has a specific activity at 37° C. which is at least 125% of the corresponding wild type reverse transcriptase.
17. The reverse transcriptase of claim 1 , wherein the reverse transcriptase has a thermostability of at least 1.5 times that of the corresponding wild type reverse transcriptase, measured as residual activity at 37° C. following treatment at 50° C. for 5 minutes.
18. A polynucleotide encoding the reverse transcriptase according to claim 1 .