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Composition and method for determining enzyme activity of M-MLV reverse transcriptase

A technology of reverse transcriptase and assay method, which is applied in the field of M-MLV reverse transcriptase enzyme activity assay composition, can solve the problems of large human factors, aerosol pollution, and long time consumption, and achieve simple assay operation and good repeatability , the effect of simple operation

Pending Publication Date: 2022-04-29
苏州译酶生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Disadvantages of the existing enzyme activity determination method: large error (need to compare with the naked eye, large human factors), long time-consuming, high cost (large amount of commercial enzyme and enzyme to be tested), need to do PCR amplification before running the gel, easy to cause gas Sol pollution

Method used

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  • Composition and method for determining enzyme activity of M-MLV reverse transcriptase
  • Composition and method for determining enzyme activity of M-MLV reverse transcriptase
  • Composition and method for determining enzyme activity of M-MLV reverse transcriptase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] SYBR dye relative enzyme activity determination process:

[0136] 1. Dilute different commercial enzymes, take 1ul each time and mix with the Mix in step 2.

[0137]

[0138] 2. Mix preparation

[0139]

[0140]

[0141] 4. Data processing, see Figure 5 .

[0142]

[0143] 4. Experimental analysis

[0144] SYBR dye tests the enzyme activity of M-MLV enzyme, can establish standard curve and R 2 Can reach 0.99

Embodiment 2

[0146] 1. Dilute Oligreen dye to 1X

[0147] 2. RNase A enzyme preparation

[0148] (1) Weigh 100mg of RNase A powder (using Gao Bingbing's balance), and put it into a 50ml centrifuge tube;

[0149] (2) Add 100uL Tris-HCl(1M), 50uLNaCl(3M), add 7mlNF water to dissolve RNase A powder;

[0150] (3) Adjust the pH value of the solution to 7.5 with HCl and NaOH, then dilute to 10ml with water, and mix the solution thoroughly;

[0151] (4) Boil in a metal bath at 100°C for 15 minutes, and slowly drop to room temperature (flocculent formation occurs);

[0152] (5) Centrifuge the RNase A solution cooled to room temperature at 3500rmp for 5min, filter it with a 0.22um filter element, and store it at -20°C.

[0153] 3. Mix preparation

[0154] TE addition amount / uL 9.27 9.02 8.77 8.27 7.77 293T-RNA amount / uL 0.73 0.73 0.73 0.73 0.73 RNase A enzyme amount / uL 0 0.25 0.5 1 1.5 Oligreen amount / uL 10 10 10 10 10

[0155] 4. React on a mic...

Embodiment 3

[0164] 1. Dilute Oligreen dye to 1X

[0165] 2. Commercial enzyme (Maxima H Minus Reverse Transcriptase) dilution

[0166]

[0167] 3. Mix preparation

[0168]

[0169]

[0170] 4. Perform amplification on the microplate reader and instrument, (42°C, 1min) for 20min

[0171] 5. Experimental map, see in details Figure 8

[0172] 6. For data processing, see Figure 9

[0173]

[0174] 7. Experimental analysis

[0175] (1) There is a background signal in the system, and when the amount of enzyme added is 0, Oligreen may combine with single strands such as RNA and primers to produce fluorescence;

[0176] (2) The more MMLV enzyme added in the system, the lower the fluorescence signal;

[0177] (3) When Oligreen dye detects M-MLV enzyme, there is a change in fluorescence signal, which can be used to test the enzyme activity.

[0178] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or ope...

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Abstract

The invention discloses an enzyme activity determination composition of M-MLV reverse transcriptase and a determination method. A fluorescent quantitative method is adopted for determining the activity of the M-MLV reverse transcriptase, and isothermal extension reaction is carried out under the condition of 37-42 DEG C. The invention also discloses a method for determining the enzyme activity of the M-MLV reverse transcriptase. According to the method, the SYBR dye is combined with the cDNA double strand to emit fluorescence, the Oligreen dye is combined with the single strand to emit fluorescence, a reaction system for determining the enzyme activity is designed, the principle that the fluorescent dye is combined with the DNA to emit fluorescence is utilized, the enzyme activity determination method of the M-MLV reverse transcriptase is designed, and compared with a traditional determination method, the time consumption is short, the operation is simple, and the repeatability is good.

Description

technical field [0001] The invention relates to the technical field of RNA reverse transcriptase, in particular to an M-MLV reverse transcriptase enzyme activity assay composition and a assay method thereof. Background technique [0002] Moloney murine leukemia virus (Moloney murine leukeminvirus) reverse transcriptase (referred to as M-MLVRTase) reverse transcriptase This enzyme has been genetically changed, that is, the ribonuclease H activity associated with it (a specialized An enzyme that cleaves an RNA molecule on a hybrid strand of DNA and RNA). This enzyme is used for first-strand synthesis of cDNA and extension of primers. This enzyme requires magnesium ions or manganese ions as cofactors. When mRNA is used as a template, single-stranded DNA (ssDNA) is first synthesized, and then under the action of reverse transcriptase and DNA polymerase I, single-stranded DNA is synthesized as a template. Hairpin" double-stranded DNA (dsDNA), and then cut into two single-strand...

Claims

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Application Information

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IPC IPC(8): C12Q1/48G01N21/64
CPCC12Q1/48G01N21/6428G01N2021/6439
Inventor 赵春艳张惠丹
Owner 苏州译酶生物科技有限公司
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