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
CN104293883AInactive Publication Date: 2015-01-21VAZYME BIOTECH NANJING
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Patent Information

Authority / Receiving Office
CN Β· China
Patent Type
Applications(China)
Current Assignee / Owner
VAZYME BIOTECH NANJING
Publication Date
2015-01-21
Estimated Expiration
Not applicable Β· inactive patent

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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.
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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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