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Researchers Submit Patent Application, "Characterization of Thermostable Dna Polymerase", for Approval

July 14, 2014



By a News Reporter-Staff News Editor at Biotech Business Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventor Jung, Laura (Oakland, CA), filed on December 18, 2013, was made available online on July 3, 2014 (see also Biotechnology Companies).

The patent's assignee is Roche Molecular Systems, Inc.

News editors obtained the following quote from the background information supplied by the inventors: "The polymerase chain reaction (PCR) process for amplifying nucleic acid sequences is well known in the art and disclosed in, for example, U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,965,188, each incorporated herein by reference. In each cycle of a PCR amplification, a double-stranded target sequence is denatured, primers are annealed to each strand of the denatured target sequence, and the primers are extended (elongated) by the action of a DNA polymerase. These steps can be summarized as denaturing, annealing and elongation step, respectively. Under the elevated temperatures used in a typical PCR, the primers hybridize only to the intended target sequence. However, amplification reaction mixtures are typically assembled at room temperature, well below the hybridization temperature of the primers. Under such less stringent conditions the primers may bind to only partially complementary sequences or other primers and initiate the synthesis of undesired extension products. These undesired extension products are amplified along with the target sequence and this amplification competes with the amplification of the desired target sequence decreasing the efficiency of specific amplification. Several methods are known in the art to increase efficiency and decrease unspecific amplification during PCR reactions. For instance, hot-start methods were initially carried out by manually opening the reaction tube after the initial high temperature incubation step and adding the missing reagents (e.g. DNA polymerase). This method was further improved by the development of chemically modified DNA polymerase. Chemically modified DNA polymerases are reversible inactivated polymerases, which are inactivated by covalent modifications, which are located at the active site of the enzyme or which cause a conformational change rendering the enzyme inactive. The chemical modification can be reversed by heat thereby transforming the enzyme into its active form. Therefore, a preassembled reaction mix containing the DNA polymerase in its inactive form can be directly used for amplification, without the extra step of adding the enzyme after the first high temperature incubation step. Chemically modified DNA polymerases and methods of modifying the same are described in U.S. Pat. Nos. 5,773,258 and 5,677,152, which are hereby incorporated by reference in their entirety.

"High quality DNA polymerases are useful for a variety of applications such as highly complex diagnostic tests that require high efficiency and specificity of the PCR amplification performed."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "The present invention provides, inter alia, methods of characterizing DNA polymerases using mass spectrometry. In particular, the methods provided herein can be used to detect chemical lysine modifications in a DNA polymerase thereby characterizing the activity level of said DNA polymerase.

"In one aspect, a method of detecting the number of covalent lysine modifications in a lysine-modified DNA polymerase is provided. The method includes (i) detecting the mass of the lysine-modified DNA polymerase at a modification stabilizing pH; and (ii) calculating the number of lysine modifications in the lysine-modified DNA polymerase by comparing the mass of the lysine-modified DNA polymerase to a polymerase without lysine modifications, wherein the number of lysine modifications is the difference of the mass of the lysine-modified DNA polymerase and the polymerase without lysine modifications divided by the mass of the lysine modification, thereby detecting the number of lysine modifications in the DNA polymerase.

"In some embodiments, the covalent lysine modification is formed by reaction of the DNA polymerase with a modifier reagent. In other embodiments, the modifier reagent is a dicarboxylic acid anhydride. In some embodiments, the dicarboxylic acid anhydride is selected from the group consisting of maleic anhydride, citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride, exo-cis-3,6-endoxo-.DELTA.4-tetrahydrophthalic anhydride and 3,4,5,6 tetrahydrophthalic anhydride. In some embodiments, the covalent lysine modification is a citraconyl modification. In some embodiments, the covalent lysine modification is an aconitylated modification. In some embodiments, the covalent lysine modification is a 2,3-dimethylmaleated modification.

"In some embodiments, the mass of the lysine-modified DNA polymerase is a computer readable mass. In some embodiments, the mass of the polymerase without lysine modifications is constructed in silico, thereby forming a computer readable standard mass. In some embodiments, the calculating is performed on a computer.

"In some embodiments, the modification stabilizing pH is at least 8. In some embodiments, the DNA polymerase is substantially free of non-ionic detergent immediately prior to and during the detecting of step (i).

"In some embodiments, the method further includes prior to the detecting of step (i), fragmenting the lysine-modified DNA polymerase with an enzyme that fragments the polymerase at known sites within the polymerase. In some embodiments, the enzyme is trypsin. In some embodiments, the method includes detecting the mass-to-charge ratio of fragments of the lysine-modified DNA polymerase and comparing the mass of fragments of the lysine-modified DNA polymerase to the mass of potential fragments of the polymerase without lysine modifications, thereby determining the location of lysine modifications at particular lysines in the polymerase.

"In some embodiments, one or more lysine modification blocks the enzyme thereby preventing the formation of one or more fragment and/or increasing the size of a resulting fragment. In some embodiments, the method includes detecting the relative amount of fragments of the lysine-modified DNA polymerase and optionally comparing the relative amount of the fragments of the lysine-modified DNA polymerase to a predicted relative amount of potential fragments of the polymerase without lysine modifications, thereby determining the relative level of lysine modifications at particular lysines in the polymerase.

"In some embodiments, the location of lysine modification corresponds to amino acid position 540, 663, or 738 of Taq GOLD polymerase. In some embodiments, the location of lysine modification corresponds to amino acid position 542, 665, or 740 of Taq Z05 polymerase.

"In some embodiments, the method provided herein includes detecting the relative amount of modification at the amino acid position, wherein the relative amount of modification is the amount of modified lysine at the position divided by the sum of the amount of modified and unmodified lysine at the position, thereby detecting the relative amount of modification at the position.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1. Diagram of the quadrupole time-of-flight mass analyzer (from Siuzdak, G, 'The Expanding Role of Mass Spectrometry in Biotechnology').

"FIG. 2. Intact analysis of delta Z05: (A) Total ion chromatogram; (B) mass spectrum; (C) deconvoluted mass. The theoretical mass of delta Z05 is 61,076.04. The mass obtained by deconvolution of the mass spectrum was 60,874.91, which is consistent with the loss of the N-terminal methionine and alanine residues, within 18 ppm (1.11 amu).

"FIG. 3. High pH intact analysis was used to determine a global distribution of 10-19 citraconyl modifications on delta Z05 GOLD. Each peak represents the mass of delta Z05 plus the designated number of citraconyl modifications.

"FIG. 4. Analysis of a PCR master mix modified to be compatible with LCMS. The enzyme concentration was increased and the salts, dNTPs, UNG and DMSO were excluded. Analysis of this master mix under accelerated storage conditions indicated a significant loss of the citraconyl modifications at elevated temperatures. Storage at 4.degree. C. showed a net loss of two modifications after four months.

"FIG. 5. Map of lysine residues in Taq GOLD and Z05 GOLD with the percentage of citraconyl modifications.

"FIG. 6. Comparison of lysine residues in the DNA polymerase domains of Taq GOLD and Z05 GOLD. The Z05 GOLD has a net of five additional lysines which are all modified to some extent. The K540, K663 and K738 in Taq and their equivalent in Z05 are all heavily modified. A molecular model of Taq lacking the exonuclease domain (front view) indicates that they are in key positions."

For additional information on this patent application, see: Jung, Laura. Characterization of Thermostable Dna Polymerase. Filed December 18, 2013 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3127&p=63&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: Lysine, Peptides, Proteins, Anhydrides, Polymerase, DNA Research, Basic Amino Acids, Organic Chemicals, Diamino Amino Acids, Enzymes and Coenzymes, Essential Amino Acids, Biotechnology Companies, Roche Molecular Systems Inc.

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Source: Biotech Business Week


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