US 2024/0392264 A1 · Taq DNA Polymerase Variants with Increased Reverse Transcriptase Activity

Abstract

Taq DNA polymerase mutants exhibiting reverse transcriptase activity compared to wild type polymerase were engineered, characterized, and selected via polymerase chain reactions visualized via electrophoresis on agarose gels. Initial screening was followed up with probe-based qualitative, real-time PCR (qPCR) with a typical reverse transcription cycling protocol to detect specified ribonucleic acid (RNA) target sequences. The engineered variants can render robust cDNA from RNA target substrates and amplify that cDNA under standard reaction conditions without the assistance of added reverse transcriptase enzymes.

A mutant Taq polymerase comprising:

one or more of the following amino acid mutations shown in the following even-numbered sequences, and wherein the remainder of the mutant polymerase has at least 80% amino acid sequence identity with wild type Taq polymerase, as shown in SEQ ID NO:
2 but wherein wild type Taq polymerase does not include the 6-membered histidine tag at its C-terminus and the six immediately preceding Glycine and Serine amino acids:
SEQ ID NO:
E9K (SEQ ID NO:
3;
SEQ ID NO:
4), L15S (SEQ ID NO:
5;
SEQ ID NO:
6), D18R (SEQ ID NO:
7;
SEQ ID NO:
8), H20E (SEQ ID NO:
9;
SEQ ID NO:
10), H21E (SEQ ID NO:
11;
SEQ ID NO:
12), K31E (SEQ ID NO:
13;
SEQ ID NO:
14), R37D (SEQ ID NO:
15;
SEQ ID NO:
16), F66A (SEQ ID NO:
17;
SEQ ID NO:
18), K82E (SEQ ID NO:
19;
SEQ ID NO:
20), A83F (SEQ ID NO:
21;
SEQ ID NO:
22), R85D (SEQ ID NO:
23;
SEQ ID NO:
24), P87G (SEQ ID NO:
25;
SEQ ID NO:
26), P89G (SEQ ID NO:
27;
SEQ ID NO:
28), E90K (SEQ ID NO:
29;
SEQ ID NO:
30), F92A (SEQ ID NO:
31;
SEQ ID NO:
32), 199S (SEQ ID NO:
33;
SEQ ID NO:
34), E101K (SEQ ID NO:
35;
SEQ ID NO:
36), L102S (SEQ ID NO:
37;
SEQ ID NO:
38), L108S (SEQ ID NO:
39;
SEQ ID NO:
40), A109F (SEQ ID NO:
41;
SEQ ID NO:
42), R110D (SEQ ID NO:
43;
SEQ ID NO:
44), G115P (SEQ ID NO:
45;
SEQ ID NO:
46., S124I (SEQ ID NO:
47;
SEQ ID NO:
48), 1138S (SEQ ID NO:
49;
SEQ ID NO:
50), V155S (SEQ ID NO:
51;
SEQ ID NO:
52), L156S (SEQ ID NO:
53;
SEQ ID NO:
54), H157E (SEQ ID NO:
55;
SEQ ID NO:
56), T164I (SEQ ID NO:
57;
SEQ ID NO:
58), L168S (SEQ ID NO:
59;
SEQ ID NO:
60), R183D (SEQ ID NO:
61;
SEQ ID NO:
62), G187P (SEQ ID NO:
63;
SEQ ID NO:
64), E189A (SEQ ID NO:
65;
SEQ ID NO:
66), E189I (SEQ ID NO:
67;
SEQ ID NO:
68), E189K (SEQ ID NO:
69;
SEQ ID NO:
70), E189S (SEQ ID NO:
71;
SEQ ID NO:
72), K202E (SEQ ID NO:
73;
SEQ ID NO:
74), E230A (SEQ ID NO:
75;
SEQ ID NO:
76), E230C (SEQ ID NO:
77;
SEQ ID NO:
78), E230M (SEQ ID NO:
79;
SEQ ID NO:
80), E230Q (SEQ ID NO:
81;
SEQ ID NO:
82), E230V (SEQ ID NO:
83;
SEQ ID NO:
84), L233S (SEQ ID NO:
85;
SEQ ID NO:
86), H235E (SEQ ID NO:
87;
SEQ ID NO:
88), M236S (SEQ ID NO:
89;
SEQ ID NO:
90), D237R (SEQ ID NO:
91;
SEQ ID NO:
92), D244R (SEQ ID NO:
93;
SEQ ID NO:
94), K247E (SEQ ID NO:
95;
SEQ ID NO:
96), L281S (SEQ ID NO:
97;
SEQ ID NO:
98), P291G (SEQ ID NO:
99;
SEQ ID NO:
100), L294S (SEQ ID NO:
101;
SEQ ID NO:
102), V310S (SEQ ID NO:
103;
SEQ ID NO:
104), K314E (SEQ ID NO:
105;
SEQ ID NO:
106), R349D (SEQ ID NO:
107;
SEQ ID NO:
108), L379S (SEQ ID NO:
109;
SEQ ID NO:
110), S383I (SEQ ID NO:
111;
SEQ ID NO:
112), Y394A (SEQ ID NO:
113;
SEQ ID NO:
114), G395P (SEQ ID NO:
115;
SEQ ID NO:
116), A442F (SEQ ID NO:
117;
SEQ ID NO:
118), E507G (SEQ ID NO:
119;
SEQ ID NO:
120), E507I (SEQ ID NO:
121;
SEQ ID NO:
122), E507L (SEQ ID NO:
123;
SEQ ID NO:
124), E537W (SEQ ID NO:
125;
SEQ ID NO:
126), T544I (SEQ ID NO:
127;
SEQ ID NO:
128), P550G (SEQ ID NO:
129;
SEQ ID NO:
130), D578F (SEQ ID NO:
131;
SEQ ID NO:
132., D578R (SEQ ID NO:
133;
SEQ ID NO:
134), D578T (SEQ ID NO:
135;
SEQ ID NO:
136., D578Y (SEQ ID NO:
137;
SEQ ID NO:
138), D732A (SEQ ID NO:
139;
SEQ ID NO:
140., D732F (SEQ ID NO:
141;
SEQ ID NO:
142), D732G (SEQ ID NO:
143;
SEQ ID NO:
144., D732I (SEQ ID NO:
145;
SEQ ID NO:
146), D732P (SEQ ID NO:
147;
SEQ ID NO:
148), D732Q (SEQ ID NO:
149;
SEQ ID NO:
150), D732S (SEQ ID NO:
151;
SEQ ID NO:
152), E742A (SEQ ID NO:
153;
SEQ ID NO:
154), E742G (SEQ ID NO:
155;
SEQ ID NO:
156), E742H (SEQ ID NO:
157;
SEQ ID NO:
158), E742I (SEQ ID NO:
159;
SEQ ID NO:
160), E742M (SEQ ID NO:
161;
SEQ ID NO:
162), E742P (SEQ ID NO:
163;
SEQ ID NO:
164), E742S (SEQ ID NO:
165;
SEQ ID NO:
166), E39K/E189K (SEQ ID NO:
167;
SEQ ID NO:
168., E39K/E230K (SEQ ID NO:
169;
SEQ ID NO:
170), E39K/E520K (SEQ ID NO:
171;
SEQ ID NO:
172), E39K/E537K (SEQ ID NO:
173;
SEQ ID NO:
174), E39K/D578R (SEQ ID NO:
175;
SEQ ID NO:
176), E39K/D732R (SEQ ID NO:
177;
SEQ ID NO:
178), E39K/E742K (SEQ ID NO:
179;
SEQ ID NO:
180), G46D/E189K (SEQ ID NO:
181;
SEQ ID NO:
182), G46D/E230K (SEQ ID NO:
183;
SEQ ID NO:
184), G46D/N384R (SEQ ID NO:
185;
SEQ ID NO:
186), G46D/D578R (SEQ ID NO:
187;
SEQ ID NO:
188), E189K/E230K (SEQ ID NO:
189;
SEQ ID NO:
190), E189K/E520K (SEQ ID NO:
191;
SEQ ID NO:
192), E189K/E537K (SEQ ID NO:
193;
SEQ ID NO:
194), E189K/D578R (SEQ ID NO:
195;
SEQ ID NO:
196), E189K/D732R (SEQ ID NO:
197;
SEQ ID NO:
198), E189K/E742K (SEQ ID NO:
199;
SEQ ID NO:
200), E230K/E520K (SEQ ID NO:
201;
SEQ ID NO:
202), E230K/E537K (SEQ ID NO:
203;
SEQ ID NO:
204), E230K/D578R (SEQ ID NO:
205;
SEQ ID NO:
206), E230K/D732R (SEQ ID NO:
207;
SEQ ID NO:
208), E230K/E742K (SEQ ID NO:
209;
SEQ ID NO:
210), E520K/E537K (SEQ ID NO:
211;
SEQ ID NO:
212), E520K/D578R (SEQ ID NO:
213;
SEQ ID NO:
214), E520K/D732R (SEQ ID NO:
215;
SEQ ID NO:
216), E520K/E742K (SEQ ID NO:
217;
SEQ ID NO:
218), E537K/D578R (SEQ ID NO:
219;
SEQ ID NO:
220), E537K/D732R (SEQ ID NO:
221;
SEQ ID NO:
222), E537K/E742K (SEQ ID NO:
223;
SEQ ID NO:
224), D578R/D732R (SEQ ID NO:
225;
SEQ ID NO:
226), D578R/E742K (SEQ ID NO:
227;
SEQ ID NO:
228), D732R/E742K (SEQ ID NO:
229;
SEQ ID NO:
230), E39K/E230K/E742K. R46D/E178K/F667Y. GR46D/E189K/E230K (SEQ ID NO:
231;
SEQ ID NO:
232), GR46D/E189K/D578R (SEQ ID NO:
233;
SEQ ID NO:
234), GR46D/E189K/F667Y (SEQ ID NO:
235;
SEQ ID NO:
236), GR46D/E189K/D732R (SEQ ID NO:
237;
SEQ ID NO:
238), GR46D/E230K/F667Y (SEQ ID NO:
239;
SEQ ID NO:
240), GR46D/E230K/D732R (SEQ ID NO:
241;
SEQ ID NO:
242), GR46D/N384R/F667Y (SEQ ID NO:
243;
SEQ ID NO:
244), GR46D/D578R/F667Y (SEQ ID NO:
245;
SEQ ID NO:
246), E189K/E230K/E520K (SEQ ID NO:
247;
SEQ ID NO:
248), E189K/E230K/E537K (SEQ ID NO:
249;
SEQ ID NO:
250), E189K/E230K/D578R (SEQ ID NO:
251;
SEQ ID NO:
252), E189K/E230K/D732R (SEQ ID NO:
253;
SEQ ID NO:
254), E189K/E230K/E742K (SEQ ID NO:
255;
SEQ ID NO:
256), E189K/E520K/E537K (SEQ ID NO:
257;
SEQ ID NO:
258), E189K/E520K/D578R (SEQ ID NO:
259;
SEQ ID NO:
260), E189K/E520K/D732R (SEQ ID NO:
261;
SEQ ID NO:
262), E189K/E520K/E742K (SEQ ID NO:
263;
SEQ ID NO:
264), E189K/E537K/D578R (SEQ ID NO:
265;
SEQ ID NO:
266), E189K/E537K/D732R (SEQ ID NO:
267;
SEQ ID NO:
268), E189K/E537K/E742K (SEQ ID NO:
269;
SEQ ID NO:
270), E189K/D578R/D732R (SEQ ID NO:
271;
SEQ ID NO:
272), E189K/D578R/E742K (SEQ ID NO:
273;
SEQ ID NO:
274), E189K/D732R/E742K (SEQ ID NO:
275;
SEQ ID NO:
276), E230K/E520K/E537K (SEQ ID NO:
277;
SEQ ID NO:
278), E230K/E520K/D578R (SEQ ID NO:
279;
SEQ ID NO:
280), E230K/E520K/D732R (SEQ ID NO:
281;
SEQ ID NO:
282), E230K/E520K/E742K (SEQ ID NO:
283;
SEQ ID NO:
284), E230K/E537K/D578R (SEQ ID NO:
285;
SEQ ID NO:
286), E230K/E537K/D732R (SEQ ID NO:
287;
SEQ ID NO:
288), E230K/D578R/D732RE230K/D578K/D732R (SEQ ID NO:
289;
SEQ ID NO:
290), E230K/D578R/D732R (SEQ ID NO:
289;
SEQ ID NO:
290), E230K/D732R/E742K (SEQ ID NO:
291;
SEQ ID NO:
292) E230K/D578R/E742K, E230K/D732R/E742K (SEQ ID NO:
291;
SEQ ID NO:
292), E520K/E537K/D578R (SEQ ID NO:
293;
SEQ ID NO:
294), E520K/E537K/D732R (SEQ ID NO:
295;
SEQ ID NO:
296), E520K/D578R/D732R (SEQ ID NO:
297;
SEQ ID NO:
298), E520K/D732R/E742K (SEQ ID NO:
299;
SEQ ID NO:
300), E537K/D578R/D732R (SEQ ID NO:
301;
SEQ ID NO:
302), E537K/D578R/E742K (SEQ ID NO:
303;
SEQ ID NO:
304), E537K/D732R/E742K (SEQ ID NO:
305;
SEQ ID NO:
306), D578R/D732R/E742K (SEQ ID NO:
307;
SEQ ID NO:
308), G46D/E189K/E230K/F667Y (SEQ ID NO:
309;
SEQ ID NO:
310), and G46D/E189K/D578R/F667Y (SEQ ID NO:
311;
SEQ ID NO:
312).

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Taq DNA Polymerase Variants with Increased Reverse Transcriptase Activity

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Taq DNA Polymerase Variants with Increased Reverse Transcriptase Activity

Life of this patent

Abstract

Claims14