Mutant polymerases with fast elongating activity

a technology of mutant polymerases and elongating activity, which is applied in the direction of transferases, enzymology, organic chemistry, etc., can solve the problems of affecting the effect of elongation, blockage amplification, and time-consuming pre-treatment steps of blood samples

Inactive Publication Date: 2013-09-26
DNA POLYMERASE TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for quickly amplifying DNA from a whole blood sample using a special cocktail of enzymes. The blood sample is layered beneath the enzymes to avoid mixing and the enzymes are activated only when the sample reaches a specific temperature. This helps to initiate amplification quickly and allows for more efficient use of the enzymes. The patent also includes a method for preparing the reaction cocktail by layering two components, one of which includes the essential constituent required for amplification. Overall, this invention provides a faster and more efficient way to amplify DNA from blood samples.

Problems solved by technology

A major problem with diagnostic and forensic techniques based on PCR is the false-negative reactions or low sensitivity caused by inhibitory substances that interfere with PCR (1, 2, 3).
However, heating of IgG together with target DNA at 95° C. was found to block amplification.
These pre-treatment steps of the blood samples are generally time-consuming, labor-intensive, and can be sample specific.
The guanidinium thiocyanate method for DNA isolation is not suitable for reliable detection of Mycobacterium tuberculosis in clinical samples.
In addition, some the above steps carry a risk of target DNA losses and are not suitable for automation.
Moreover, even commercial kits specially formulated for DNA purification from blood samples such as QIAmp or GeneReleaser are not always satisfactory.
The reason is due to an incomplete removal of Taq inhibitors, which can result in false-negative results.
The objective of achieving specificity of amplification reactions for samples containing whole blood is further complicated by two types of unwanted DNA synthesis reactions that occur during PCR.
Both types of side-reactions are frequently competitive with the desired target and can lead to impure product or failed amplification.
This is particularly problematic for PCR assays containing a low copy number of the nucleic acid template target, wherein the PCR conditions are modified to include a greater number of amplification cycles to achieve an adequate yield of the desired amplification product.
This is only an issue if the template is contaminated with single-stranded nucleic acid or if the template is single-stranded, which is the case if the DNA preparation has been subjected to melting conditions during its isolation.
These so-modified primers are able to anneal to the nucleic acid target; however, they do not serve as primers for complementary strand synthesis due to the presence of mismatched nucleotides at the site of elongation between the 3′ end of the primer and the desired target.
This problem can often be reduced or avoided by careful primer design, and it is more of a problem with multiplex PCR, since there is more opportunity for accidental homology among multiple pairs of primers.
At this temperature the primers can presumably not form stable duplexes with themselves or at unwanted template sequences.
Typical hot start PCR procedures are not only labor-intensive, they expose the PCR reactions to contamination with each other and with molecules that have been previously amplified in the thermal cycler machine.
A great drawback to the wax method comes after the PCR cycling is complete, and the product must be withdrawn for analysis.
The wax then tends to plug the pipette tip, greatly adding to the time and effort of reaction analysis.
The antibodies so far developed for this method must be used in 10-fold molar excess and are expensive.
The nature of the inactivation is proprietary, but the inactivation is reversible by heating the polymerase at 95° C. This method may be even more convenient than the other methods, but it has at least one current disadvantage: the time for reactivation is about 10 minutes at 95° C. This procedure is incompatible with long PCR applications, as this treatment would excessively depurinate nucleic acid targets longer than a few kb.

Method used

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  • Mutant polymerases with fast elongating activity
  • Mutant polymerases with fast elongating activity
  • Mutant polymerases with fast elongating activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

Screening of Mutagenized Klentaq Clones for Blood-Resistant Mutant Enzyme Activity

[0122]In order to functionally characterize new mutants, it is desirable to produce highly-purified enzyme from expression systems. The procedure, which included PEI treatment, BioRex-70 chromatography, and Heparin-Agarose chromatography, yielded DNA-free and nuclease-free Klentaq enzyme purified to homogeneity, as judged by a single band in Coomassie stained protein gel (23). The same purification procedure also worked very well for purification of cold sensitive Klentaq mutants (23). This procedure was readily adaptable to accommodate purification of mutant polymerases that display unusual features such as changed affinity and elution profile on a particular chromatography resin. The efficiency of each step in the purification scheme was monitored easily by a standard DNA incorporation assay.

[0123]The amplification activity of the obtained mutant enzymes were extensively evaluated in PCR amplificatio...

example 2

Full-Length Taq DNA Polymerase Mutants Display Blood-Resistant Activity

[0127]Importantly, the amino acid changes responsible for the blood-resistant phenotype of the Klentaq, were also sufficient to render the full-length Taq blood-resistant when these amino acid changes were incorporated into the full-length gene. For example, the amino acid changes of KT-10 and KT-12 mutants were incorporated into the full-length Taq gene to generate the analogous Taq-mutants FL-10 and FL-12. As shown in FIG. 3A (for FL-10) and FIG. 3B (for FL-12), both full-length Taq mutants exhibited very high resistance to blood inhibition, and successfully amplified the endogenous human Dystrophin and CCR5 genes in homogeneous PCR solutions containing 20% blood. The observed high blood resistance of these mutants reflects dramatic change in the property of the Taq enzyme, considering the fact that the wild-type Taq is typically inactivated in homogeneous PCR assay solutions containing as little as 0.1-0.5% wh...

example 3

Mutagenized Klentaq Mutants with a Faster DNA Elongation Rate

[0128]The screening factor here is to simply shorten the DNA extension step of the PCR cycle beyond the point where the wild-type or prior art enzyme stops working. In the case when wild-type Klentaq amplified its own gene, the amplification efficiency was significantly lower at 60 seconds extension step (FIG. 4A, lane 1 at 1 min). Additional tests with discrete extension times showed that the Klentaq polymerase did not display amplification activity in PCR assays performed under conditions that employ an extension time of about 50 sec or less (e.g., see FIG. 4A, lane 1 at 30 sec and 20 sec). On the other hand, mutant Klentaq clone KT-7 displayed amplification activity with the same target in PCR assays under conditions having an extension step of as little as about 12 sec. (FIG. 4B, lower panel). For the evaluation of fast-elongating mutants, extension times in the PCR cycle not exceeding 20 sec per 2 kb amplicon were use...

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Abstract

An isolated polypeptide having fast elongating polymerase activity. Also provided are kits containing the isolated polypeptide and isolated polynucleotides encoding the isolated polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is a Continuation of U.S. application Ser. No. 12 / 330,201 filed on 8 Dec. 2008, which is a Divisional of U.S. application Ser. No. 11 / 005,559 filed on 6 Dec. 2004 issued as U.S. Pat. No. 7,462,475 on 9 Dec. 2008, which is a Continuation-in-part of U.S. application Ser. No. 10 / 850,816 filed 20 May 2004, each incorporated herein by reference in their entirety to the extent permitted by law, and claims benefit of priority therefrom.INCORPORATION BY REFERENCE OF SEQUENCE LISTING[0002]The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and / or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND[0003]The polymera...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12N9/12C12P19/34C12Q1/68
CPCC12Q1/686C12N9/1252C12Q2549/101C12Q2521/101
InventorKERMEKCHIEV, MILKO B.BARNES, WAYNE M.
OwnerDNA POLYMERASE TECH