	US 12,234,488 B2
	Mutant reverse transcriptase with increased thermal stability as well as products, methods and uses involving the same
Christian Helmut Bell, Basel (CH); Harald Sobek, Biberach (DE); Heiko Walch, Munich (DE); Kiyoshi Yasukawa, Koyoto (JP); and Misato Baba, Hyto (JP)
Assigned to Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed by Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed on Dec. 20, 2022, as Appl. No. 18/068,739.
Application 18/068,739 is a division of application No. 16/599,407, filed on Oct. 11, 2019, granted, now 11,618,892.
Application 16/599,407 is a continuation of application No. PCT/EP2018/059170, filed on Apr. 10, 2018.
Claims priority of application No. 17000621 (EP), filed on Apr. 11, 2017.
Prior Publication US 2023/0203460 A1, Jun. 29, 2023
Int. Cl. C12P 19/34 (2006.01); C12N 9/12 (2006.01); C12Q 1/68 (2018.01)

CPC C12N 9/1276 (2013.01) [C12P 19/34 (2013.01); C12Q 1/68 (2013.01); C12Y 207/07049 (2013.01)]

4 Claims

1. A method for reverse transcription of a mRNA, the method comprising synthesizing a cDNA from the mRNA using a mutant reverse transcriptase (RT), the mutant RT consisting of

i) an amino acid sequence that has only six amino acid substitutions of SEQ ID NO: 1, wherein, in SEQ ID NO:1,

Ala at position 32 is substituted with Val (A32V);

Leu at position 72 is substituted with Arg (L72R);

Glu at position 286 is substituted with Arg (E286R);

Glu at position 302 is substituted with Lys (E302K);

Trp at position 388 is substituted with Arg (W388R); and

Leu at position 435 is substituted with Arg (L435R),

wherein the mutant RT exhibits a reverse transcriptase activity, and wherein the mutant RT also has an increased thermal stability relative to a mutant MM3, wherein the mutant MM3 has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 and only has three amino acid substitutions of SEQ ID NO: 1, wherein, in SEQ ID NO:1, Glu at position 286 is substituted with Arg (E286R), Glu at position 302 is substituted with Lys (E302K) and Leu at position 435 is substituted with Arg (L435R).