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 comprising SEQ ID NO.: 25 with at least one mutation at at least one of the following amino acid positions: Q374, W388, R390, Q430, D449, N479, and E673, the isolated reverse transcriptase having increased thermostability.
2. The reverse transcriptase according to claim 1 , having one or more of the following mutations selected from the group consisting of Q374R; W388R; R390W; Q430R; D449G or A; N479D; and E673G or K.
3. The reverse transcriptase of claim 1 further comprising at least one mutation at an amino acid position selected from the group consisting of E5, M39, I49, M66, Q91, P130, L139, I179, D200, Q221, Q237, T287, A307, T330, L333, Y344, A502, H594, L603, E607, A644, N649, D653, K658, and L671.
4. The reverse transcriptase according to claim 3 , having one or more of the following mutations selected from the group consisting of E5K; M39V or L; I49V or T; M66L; Q91R or L; P130S; L139P; I179T or V; D200N, A or G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; H594R or Q; L603W or M; E607K, G or A; A644V or T; N649S; D653G, A, H or V; K658R or Q; and L671P.
5. The reverse transcriptase of claim 3 further comprising at least one mutation at an amino acid position selected from the group consisting of D524, L528, and H634.
6. The reverse transcriptase of claim 5 comprising one or more of the following mutations selected from the group consisting of D524A, L528I, and H634Y and having increased thermostability.
7. The reverse transcriptase according to claim 3 having the mutation H594K.
8. An isolated reverse transcriptase comprising SEQ ID NO.: 25 with at least one mutation selected from the group consisting of E5K; M39V or L; I49V or T; M66L; Q91R or L; P130S; L139P; I179T or V; D200N, A or G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; D524A; L528I; H594R, K or Q; L603W or M; E607K, G or A; H634Y; A644V or T; N649S; D653G, A, H or V; K658R or Q; and L671P, the isolated reverse transcriptase having increased thermostability.