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Method and kit for measuring terminal transferase activity

A terminal transferase and kit technology, applied in the field of molecular biology, can solve problems such as difficult to achieve high throughput, automation, radioactive pollution, long cycle, etc., and achieve simple and fast operation steps, no radioactive pollution, and low reagent cost Effect

Active Publication Date: 2021-10-08
GUANGDONG FAPON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic method for measuring terminal transferase activity is the radioactive substrate incorporation method. Although this method has high sensitivity, it is limited by the quality of the markers, and it is easy to produce radioactive contamination during the operation. There are many steps and a long cycle, making it difficult to achieve high throughput. ,automation

Method used

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  • Method and kit for measuring terminal transferase activity
  • Method and kit for measuring terminal transferase activity
  • Method and kit for measuring terminal transferase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] 1. Establishment of a method for the determination of terminal transferase activity by fluorescence method

[0063] 1.1 Preparation of PCR template

[0064] Template to add Poly A tail:

[0065] 5'-ACTACTTCCTTAAGATCATCCAACTATTGCCAGCAGGTGTTCGACAATGGCGATGATTCCGAAATGTTTCATTGTGGGAGCAGACAATG-3' (SEQ ID NO: 1).

[0066] Template with Poly T-tail:

[0067] 5'-TCCCTTCGCGGGAAGGCTGTGGTGCTGATGGG (SEQ ID NO:2)-TTTTTTTTTTTTTTTTTTTTTTTT-3'.

[0068] Add Poly A tail reaction system:

[0069]

[0070]

[0071] Reaction conditions for adding Poly A tail:

[0072] temperature time 37℃ 15min 65℃ 10min 4℃ ∞

[0073] 1.2 Fluorescence quantitative reaction system:

[0074] The forward primer is: 5'-CTACTTCCTTAAGATCATCCAAC-3' (SEQ ID NO:3);

[0075] The reverse primer is 5'-CTTCGCGGGAAGGCTGTGGT-3' (SEQ ID NO: 4);

[0076] The probe was (FAM) 5'-CCAGCAGGTGTTCGACAATGGC-3' (SEQ ID NO: 5) (BHQ1).

[0077] Fluorescence quantitative system components ...

Embodiment 2

[0093] For the drawing of standard curves of terminal transferase activity-CT values ​​from different sources and activity detection, we selected two commercial terminal transferases and expressed and purified terminal transferases by ourselves. Self-made terminal transferase was cloned from calf thymus terminal transferase gene, cloned into pET28a expression vector and transformed into Escherichia coli BL21 for expression. The expressed product was purified to a purity of about 98% by conventional chromatography. All terminal transferase 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 dTTP into the acid-insoluble precipitate within 1 hour. According to the method of Example 1, the data table of terminal transferase activity-CT value was drawn. The result is as follows:

[0094] Dilution factor of control standar...

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Abstract

The invention relates to a method and kit for measuring terminal transferase (TdT) activity. The method comprises: using TdT to add PolydN to the 3' end of the first single-stranded linear DNA; using the second single-stranded linear DNA as a second single-stranded template, the 3' end of the second single-stranded linear DNA having PolydN ', the second single-stranded template and the first single-stranded template are complementary paired only through the N-N' part; after complementing the complementary DNA, amplify and quantitatively detect the amplified product, and according to the amplified product The corresponding relationship between the content and the TdT enzyme activity determines the TdT activity; the upstream and downstream primers used in the amplification are respectively combined with the first single-stranded template and the second single-stranded template, and the binding part does not include N-N 'complementary regions, or both N-N' complementary regions and non-N-N' complementary regions. The method has simple and rapid operation steps, low reagent cost and high sensitivity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method and kit for measuring terminal transferase activity. Background technique [0002] Terminal deoxynucleotidyl transferase (Terminal deoxynucleotidyl transferase, TdT), also known as terminal deoxynucleotidyl transferase. Generally isolated from bovine thymus, it is a template-free DNA terminal transferase that catalyzes the incorporation of deoxynucleotides into the 3' hydroxyl end of DNA molecules. Single and double stranded DNA molecules with protruding, recessed or blunt ends can be used as TdT substrates. [0003] Terminal transferase is an important tool enzyme in genetic engineering technology. Terminal transferase catalyzes the addition of nucleotides to the 3' end of DNA molecules. Unlike most DNA polymerases, it does not require a template for its catalysis. The preferred substrate of this enzyme is the 3' overhang, but it can also add nucleotides to blunt end...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/48
CPCC12Q1/485C12Q1/6851G01N2333/9127C12Q2561/113C12Q2563/107
Inventor 蔡统聪
Owner GUANGDONG FAPON BIOTECH CO LTD