Reagent and method for improving reverse transcriptase performance

A reverse transcriptase and reagent technology, applied in the biological field, can solve problems that do not involve additive technology

Inactive Publication Date: 2013-07-03
SNOVA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Neither of the above two patents involves additive technology

Method used

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  • Reagent and method for improving reverse transcriptase performance
  • Reagent and method for improving reverse transcriptase performance
  • Reagent and method for improving reverse transcriptase performance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Wild-type M-MLV reverse transcriptase (Silo Bio, containing 1 mM DTT) was added to Reagent A described below at a volume ratio of 9:1. After mixing by pipetting for several minutes, the obtained enzyme solution was added to the B reagent described below at a volume ratio of 9:1.

[0057] Prepare A reagent according to the following formula:

[0058]

[0059]

[0060] Prepare B reagent according to the following formula:

[0061]

[0062] Taking the enzyme solution as a sample, the fluorescence real-time quantitative analysis system (Agilent, Mx3000p) and real-time quantitative PCR instrument (ABI, StepOne TM Real-Time PCR System), PCR instrument, electrophoresis instrument and other instruments, refer to the instruction manual, measure the reverse transcriptase RT activity and RNase H activity of the sample solution, and calculate the remaining activity. The results are shown in Table 1.

[0063] As can be seen from the results in Table 1, in the reagent of ...

Embodiment 2

[0067] Wild-type M-MLV reverse transcriptase (Silo Bio, containing 1 mM DTT) was added to Reagent A described below at a volume ratio of 9:1. After mixing by pipetting for several minutes, the obtained enzyme solution was added to the B reagent described below at a volume ratio of 9:1.

[0068] Prepare A reagent according to the following formula:

[0069]

[0070] Prepare B reagent according to the following formula:

[0071]

[0072] Taking the enzyme solution as a sample, the fluorescence real-time quantitative analysis system (Agilent, Mx3000p) and real-time quantitative PCR instrument (ABI, StepOne TM Real-Time PCR System), PCR instrument, electrophoresis instrument and other instruments, refer to the instruction manual, measure the RT activity and RNase H activity of the sample solution, and calculate the remaining activity. The results are shown in Table 2.

[0073] As can be seen from the results in Table 2, in the reagent of this example, after M-MLV was trea...

Embodiment 3

[0077] Wild-type M-MLV reverse transcriptase (Silo Bio, containing 1 mM DTT) was added to Reagent A described below at a volume ratio of 4:1.

[0078] Prepare A reagent according to the following formula:

[0079]

[0080] Taking the enzyme solution as a sample, the fluorescence real-time quantitative analysis system (Agilent, Mx3000p) and real-time quantitative PCR instrument (ABI, StepOne TM Real-Time PCR System), PCR instrument, electrophoresis instrument and other instruments, refer to the instruction manual, measure the RT activity and RNase H activity of the sample solution, and calculate the remaining activity. The results are shown in Table 3.

[0081] As can be seen from the results in Table 3, in the reagent of this example, after M-MLV was treated with iodoacetamide with a mother solution concentration of 25 mM (5 times the final concentration), RT activity was lost by 48%, and RNase H activity was completely lost. The test results show that the reagent in this...

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Abstract

The invention discloses a reagent and a method for improving reverse transcriptase performance. The main component of the reagent is a mercapto sealing agent, or a disulfide-bond reducing agent, or a combination of the two. The invention also discloses a method for improving reverse transcriptase performance. According to the method, the mercapto sealing agent or the disulfide-bond reducing agent or the composition of the two is introduced into the reverse transcriptase. With the reagent provided by the invention, reverse transcriptase performance can be effectively improved, such that the reverse transcriptase has higher extension capacity and higher reaction sensitivity, and higher reverse transcription temperature can be adopted. Therefore, reverse transcription difficulty caused by template RNA secondary structure is solved. With the reagent and the method, high-ratio full-length cDNA library can be constructed, and relatively long cDNA can be synthesized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a reagent capable of improving the performance of reverse transcriptase and its application method. Background technique [0002] Reverse transcriptase (Reverse transcriptase) is an enzyme that uses RNA as a template to guide deoxynucleotide triphosphates to synthesize complementary DNA (cDNA). It mainly performs the following functions: DNA polymerization dependent on RNA or DNA as a template; strand conversion; degradation RNase H function of RNA in RNA-DNA hybrids. Therefore, reverse transcriptase is often used in the construction of cDNA library. At present, AMV reverse transcriptase purified from Avian Myeloblastosis Virus (AMV) and Moloney Murine Leukemia Virus (Moloney Murine Leukemia Virus) recombinantly expressed from Escherichia coli (E.coli) M-MLV reverse transcriptase purified from M-MLV for short) is the two most commonly used reverse tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
CPCC12P19/34C12N9/1276C12N9/96C12Q1/6844C12Q2521/107C12Q2527/125
Inventor 不公告发明人
Owner SNOVA BIOTECH
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