# US 7,410,782 B2 · Thermostable enzyme promoting the fidelity of thermostable DNA polymerases—for improvement of nucleic acid synthesis and amplification in vitro

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- Number: US 7,410,782 B2
- Document ID: US7410782B2
- Status: Granted
- Kind: B2
- Application number: 11/241,116
- Filed: 2005-09-29
- Published: 2006-04-13
- Granted: 2008-08-12
- Earliest claimed: 1999-09-28
- Assignees: Roche Diagnostics GmbH; Roche Molecular Systems, Inc.
- Inventors: Waltraud Ankenbauer; Frank Laue; Harald Sobek; Michael Greif
- Web page: https://patentgrep.com/patent/US7410782B2

## CPC

- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A purified thermostable enzyme is derived form the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3′-5′ exonuclease and cleaves to produce 5′-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).

## Claims

### Claim 1

A method of preparing or amplifying DNA comprising incubating DNA with a composition comprising a first thermostable Archeoglobus fulgidus enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and a second enzyme exhibiting DNA polymerase activity and lacking proofreading activity, wherein said first thermostable Archeoglobus fulgidus enzyme is active under the incubation and temperature conditions of a polymerase chain reaction, said composition enhancing the fidelity of an amplification process in comparison to the use of the single second enzyme.

### Claim 2 (depends on 1)

The method of claim 1 wherein prematurely terminated chains are trimmed by degradation from 3′ to 5′.

### Claim 3 (depends on 1)

The method according to claim 1 wherein mismatched ends of either a primer or the growing strand are removed.

### Claim 4 (depends on 1)

The method according to claim 1 wherein dUTP instead of TTP is present in the reaction mixture.

### Claim 5 (depends on 4)

The method according to claim 4 wherein UNG is used for degradation of contaminating nucleic acids.

### Claim 6 (depends on 1)

The method according to claim 1 wherein the mixture of a
first thermostable Archeoglobus fulgidus enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and
a second enzyme exhibiting DNA polymerase activity
produces PCR products with lower error rates compared to PCR products produced by the second enzyme exhibiting DNA polymerase activity in absence of the first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity.

### Claim 7 (depends on 6)

The method of claim 6 in which the mixture of first thermostable Archeoglobus fulgidus enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity and a second enzyme exhibiting DNA polymerase activity produces PCR products of greater length compared to PCR products produced by the second enzyme exhibiting DNA polymerase activity in absence of the first thermostable enzyme exhibiting 3′-exonuclease-activity but essentially no DNA polymerase activity.

### Claim 8 (depends on 1)

The method according to claim 1 wherein PCR products with blunt ends are obtained.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first thermostable Archeoglobus fulgidus enzyme is encoded by DNA which hybridizes to a primer having a sequence as shown in SEQ ID NO: 1 and a primer having a sequence as shown in SEQ ID NO: 2.

### Claim 10 (depends on 9)

The method of claim 9, wherein the first thermostable Archeoglobus fulgidus enzyme is SEQ ID NO: 21.