Thermostable chimeric nucleic acid polymerases and uses thereof

a technology of chimeric nucleic acid and polymerases, applied in the field of molecular biology, can solve the problems of slow dna extension rate, high error rate and less desirable use of taq dna polymerase in most nucleic acid amplification applications, and achieve the effect of not losing significant enzymatic activity

Inactive Publication Date: 2014-06-05
QIAGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The chimeric polymerase maintains high enzymatic activity at elevated temperatures, reducing the need for enzyme supplementation during PCR cycles and enhancing the accuracy and efficiency of nucleic acid amplification by incorporating proofreading capabilities, thus overcoming the limitations of existing thermostable polymerases.

Problems solved by technology

Because Taq polymerase exhibits essentially no 3′-5′ exonuclease activity (i.e., proofreading capability), the error rate of Taq DNA polymerase is high compared to other DNA polymerases that possess an enzymatically active 3′-5′ exonuclease domain (Flaman et al., 1994).
Since Taq DNA polymerase does not possess an enzymatically active 3′-5′ exonuclease domain, providing a proofreading feature to the polymerase, the use of Taq DNA polymerase becomes less desirable for most nucleic acid amplification applications, e.g., for PCR sequencing protocols or amplification for protein expression, which require complete identity of replication products to the template nucleic acid.
These DNA polymerases show slower DNA extension rates and an overall lower processivity when compared to Taq DNA polymerase, however, thus rendering these naturally occurring thermostable DNA polymerases less desirable for PCR, despite their higher fidelity.
PCR protocols utilizing multiple polymerase mixtures are still prone to error, however, and require the practitioner to perform preliminary experimental trials, to determine special optimized solution conditions necessary for multiple-enzyme reaction mixtures.
They succeeded to insert a thioredoxin binding domain from T7 DNA polymerase into E. coli pol I. The inserted 76 amino acid binding domain improved polymerase binding to a template polynucleotide, thus increasing the processivity of the recombinant E. coli pol I but did not improve or provide any novel enzymatic activity to the polymerase.
(i.e., they are not thermostable), and thus are not useful for performing PCR protocols without the successive addition of fresh polymerase for each cycle.

Method used

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  • Thermostable chimeric nucleic acid polymerases and uses thereof
  • Thermostable chimeric nucleic acid polymerases and uses thereof
  • Thermostable chimeric nucleic acid polymerases and uses thereof

Examples

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example 1

Construction of a Thermostable Chimeric DNA Polymerase Gene

[0082]Chimeric thermostable DNA polymerase constructs containing enzymatically active domains from different (source) thermostable DNA polymerases were generated using recombinant DNA techniques. The 3′-5′ exonuclease domain of various thermostable polymerases were recombinantly linked to the 5′-3′ polymerase domain of Taq polymerase or Tth polymerase. The particularly preferred enzymatic domains and domain borders, described herein in detail, were selected and tested as preferred embodiments, and are not to be considered limiting in scope of the thermostable chimeric nucleic acid polymerase of the invention, or the enzymatically active domains useful therein.

[0083]Appropriate microbial strains or genomic DNA preparations, from which the enzymatically active domains used in the construction of chimeric nucleic acid polymerase were isolated, were purchased from commercial suppliers, e.g., from DSMZ GmbH (Deutsche Sammlung von...

example 1.1

Construction of a Pho / Taq Thermostable Chimeric DNA Polymerase Gene

[0091]A polynucleotide encoding the enzymatically active 3′-5′ exonuclease domain of Pho DNA polymerase was linked to a polynucleotide encoding the enzymatically active 5′-3′ polymerase domain and the nonfunctional 3′-5′ exonuclease domain of Taq DNA polymerase. A polynucleotide encoding amino acids 271-832 (SEQ ID NO:7) of Taq DNA polymerase was recombinantly linked to the 3′ end of a polynucleotide encoding amino acids 1-396 (SEQ ID NO:3) of Pho DNA polymerase following the procedures detailed in Example 1 above, producing a polynucleotide that encodes a novel Pho / Taq thermostable chimeric DNA polymerase (SEQ ID NO:8).

example 1.2

Construction of a Pwo / Taq Thermostable Chimeric DNA Polymerase Gene

[0092]A polynucleotide encoding the enzymatically active 3′-5′ exonuclease domain of Pwo DNA polymerase was linked to a polynucleotide encoding the enzymatically active 5′-3′ polymerase domain of Taq DNA polymerase. A polynucleotide encoding amino acids 271-832 (SEQ ID NO:7) of Taq DNA polymerase was recombinantly linked to the 3′ end of a polynucleotide encoding amino acids 1-396 (SEQ ID NO:4) of Pwo DNA polymerase following the procedures detailed in Example 1 above, producing a polynucleotide that encodes a novel Pwo / Taq thermostable chimeric DNA polymerase (SEQ ID NO:9).

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Abstract

Novel thermostable chimeric nucleic acid polymerases and methods for their generation and use are disclosed. It is shown that these chimeric nucleic acid polymerases, such as DNA polymerases, can be constructed using enzymatically active domains, isolated from different proteins or chemically synthesized. It is demonstrated that chimeric nucleic acid polymerases of the present invention possess the chemical and physical properties of their component domains (e.g., exonuclease activity, thermostability) and that the chimeric polymerases are thermostable.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a continuation of U.S. application Ser. No. 10 / 216,682, filed Aug. 8, 2002 (currently pending), which is a continuation of International Application No. PCT / EP01 / 01790, filed Feb. 16, 2001 (now abandoned), which is a continuation-in-part of U.S. Ser. No. 09 / 506,153, filed Feb. 17, 2000 (now abandoned), the disclosures of which are incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created Dec. 16, 2013, is named 0051—0003US2—Sequence_Listing.txt and is 84489 bytes in size.FIELD OF THE INVENTION[0003]The present invention is in the field of molecular biology. The present invention is directed to novel thermostable chimeric enzymes useful for the generation of nucleic acids, methods for making thermostable chimeric nucleic acid pol...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12P19/34C12N1/21C12N9/12C12N15/54
CPCC12P19/34C07K2319/00C12N9/1252
InventorLOFFERT, DIRKMISSEL, ANDREASKANG, JIE
OwnerQIAGEN GMBH