Production of nucleic acid
The present invention provides a process for the production of nucleic acid encoding a target protein, which 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. An isolated reverse transcriptase having an optimum activity at a temperature above 42° C., wherein the optimum activity at a temperature above 42° C. is an increased activity of the isolated reverse transcriptase compared to the activity of the isolated reverse transcriptase at 37° C., and wherein the reverse transcriptase comprises SEQ ID NO: 25 with at least one mutation selected from the group consisting of D200Q; D200V; D200 W; D200K; L603W; L603F; D653K; D653H; D200N, L603W; D200N, L603W, T330P; D200N, L603W, T330P, E607K; D200N, L603W, T330P, E607K, L139P; D200N, L603W, N479D, H594R; D200N, L603W, D653N, D524G; and D200N, L603W, D653N, D524G, T330P.
2. The reverse transcriptase according to claim 1 , wherein the optimum activity is at a temperature in the range of from 50° C. to 60° C.
3. An isolated reverse transcriptase comprising SEQ ID NO: 25 with at least one mutation selected from the group consisting of L139P, D200A, G, Q, V, W or K, L603W or F, and D653K or H.
4. An isolated reverse transcriptase of claim 1 , wherein the activity at 50° C. is at least 120% of the activity at 37° C.
5. The reverse transcriptase of claim 3 further comprising at least one mutation at an amino acid position selected from the group consisting of E5, M39, I49, M66, Q91, P130, I179, D200, Q221, Q237, T287, A307, T330, L333, Y344, Q374, W388, R390, Q430, D449, N479, A502, H594, L603, E607, A644, N649, D653, K658, L671, and E673.
6. The reverse transcriptase according to claim 5 , having at least one mutation selected from the group consisting of ESK; M39V or L; I49V or T; M66L; Q91R or L; P130S; I179T or V; D200N; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; Q374R; W388R; R390 W; Q430R; D449G or A; N479D; A502V; H594R or Q; L603M E607K, G or A; A644V or T; N649S; D653G, A or V; K658R or Q; L671P; and E673G or K.