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Method for measuring activity of terminal deoxynucleotidyl transferase

A technology for activity determination and deoxynucleoside triphosphate, applied in the field of biochemistry, can solve the problems of long cycle, many steps, difficult to achieve high-throughput, automation, etc., and achieve the effect of low cost, high sensitivity and automatic activity determination

Inactive Publication Date: 2015-01-21
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, all bioassay methods based on the "incorporation method" have the defects of many steps and a long cycle, and it is difficult to achieve high-throughput and automation

Method used

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  • Method for measuring activity of terminal deoxynucleotidyl transferase
  • Method for measuring activity of terminal deoxynucleotidyl transferase
  • Method for measuring activity of terminal deoxynucleotidyl transferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Feasibility verification of "transformation method" to measure TdT activity

[0047] Preparation of M13 template primer / complex:

[0048] M13 ssDNA: M13mp18 ssDNA (NEB);

[0049] Primer: M13 primer 5'-aagccatccgcaaaaatgacctct-3';

[0050] M13 template primer / complex: Mix M13mp18 single-stranded DNA with M13 primer at a molar ratio of 1:1, heat at 70°C for 5 minutes in annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), slowly Cool down to room temperature to anneal the template primers.

[0051] Preparation of M13 template / mismatched primer complexes:

[0052] M13 ssDNA: M13mp18 ssDNA (NEB);

[0053] Primer: M13 mismatch primer 5'-aagccatccgcaaaaatgacctct a -3' (the underline indicates the base that does not match at the end);

[0054] M13 template / mismatched primer complex: M13mp18 single-stranded DNA and M13 mismatched primer were mixed at a molar ratio of 1:1, heated at 70°C for 5 Minutes later, slowly cool down to room temperature to anneal the t...

Embodiment 2

[0070] Example 2. Drawing of TdT activity-fluorescence intensity curve and EC50 calculation

[0071] Take the log value of the TdT activity unit (U) of 2#-12# as the abscissa, and the fluorescence intensity as the ordinate, and use GraphPad Prism5 to make a nonlinear regression curve. We found that these data points can be well fitted with an inverted sigmoid curve, such as figure 2 shown.

[0072] By repeating the experiment several times and calculating the half-maximal effect concentration (EC50) of the curve, we found that the EC50 value remained stable across multiple experiments:

[0073]

[0074] Average (U) 7.02 Standard deviation SD (U) 0.049 Coefficient of Variation CV (%) 0.69

Embodiment 3

[0075] Example 3. Drawing of TdT activity-fluorescence intensity standard curves from different sources and definition of "conversion method" activity

[0076] We also selected two TdT enzymes of different species and different preparation methods, and according to the method of Example 1 and Example 2, the activity-fluorescence intensity standard curve was drawn and the EC50 was calculated. Wherein mouse thymus TdT was cloned according to (Boule et al., Mol Biotechnol. 1998 Dec;10(3):199-208) and expressed and purified in Escherichia coli; human thymus TdT was cloned according to (Deibel et al., J Biol Chem. 1979 Sep 10;254(17):8634-40) was purified from Jurkat cells. All TdT enzyme activities were determined by standard radioisotope incorporation. International unit (IU) is defined as: 1 unit (U) is defined as the amount of enzyme required to incorporate 1 nmol of isotope-labeled dATP into the acid-insoluble precipitate within 1 hour. The result is as follows:

[0077] ...

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Abstract

The invention discloses a method for measuring activity of terminal deoxynucleotidyl transferase. The method comprises the steps of: firstly, synthesizing a primer according to a single stranded template, annealing the single stranded template, then performing a 3' end tailing reaction catalyzed by TdT enzyme in the presence of one of four deoxyribonucleoside triphosphates, after the 3' end tailing reaction is completed, performing a polymerization reaction in the presence of enough DNA polymerase without 3'-5 'excision enzyme activity to generate double stranded DNA, finally measuring the relative quantity of the double stranded DNA or single stranded DNA in the reaction system after the polymerization reaction is finished, and deducing the activity degree of the Poly (A) polymerase according to the detection result, wherein the basic group at the first site on the used single stranded template in the 3' direction and the deoxyribonucleoside triphosphate used in the tailing reaction are not complementary. Compared with the existing method, the method disclosed by the invention is free of radioactive pollution, simple and quick to operate, low in reagent preparation cost and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and specifically relates to a method for measuring terminal deoxynucleotidyl transferase activity. Background technique [0002] Terminal deoxynucleotidyl transferase (terminal deoxynucleotidyl transferase, TdT) is a special DNA polymerase. TdT can catalyze the polymerization of deoxynucleoside triphosphate (dNTP) to the 3'-OH end of single-stranded or double-stranded DNA fragments one by one without relying on the template. The enzyme is specifically distributed in the thymus, bone marrow of mammals and malignant cells of certain types of leukemia patients, and can be used for diagnosis and prognosis of leukemia by measuring its activity. (Rudzka E et al., Folia Haematol Int Mag Klin Morphol Blutforsch. 1985;112(6):864-71) (Hutton JJ et al., Blood. 1982 Dec;60(6):1267-76.). Therefore, the determination of TdT activity in vivo has very important clinical diagnostic value. [0003] TdT is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
Inventor 徐晓昱王静
Owner VAZYME BIOTECH NANJING
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