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Method for promoting gene cloning efficiency

A gene and efficiency technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of complex steps, low efficiency in finding main effect factors and optimal reaction conditions, time-consuming and labor-intensive, etc., and achieve the effect of improving the success rate.

Inactive Publication Date: 2012-09-12
XIAMEN UNIV
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Problems solved by technology

However, in actual operation, some difficulties are often encountered, especially 5’-RACE, which is slightly complicated in steps and may encounter many problems
With the increasing improvement of RACE technology, various commercial RACE technology products have been launched at present. However, there are still many problems in the actual operation of RACE reaction experiments, and it is necessary to repeatedly explore the RACE reaction conditions, especially 5’-RACE
For example, for the classic 5'-RACE, it mainly includes reverse transcription, homopolymer tailing, synthesis of the second strand of cDNA and subsequent nested (nested) PCR. These consecutive enzymatic reactions Each of the steps can fail, leading to suboptimal final amplification results
Many research groups ([1] Deng Xueke, Yin Jianhua, Cao Yi. Comparison and optimization of three 5'RACE techniques [J]. Chengdu Medical College Journal. 2007, 2(1): 20-25; [2] Li Guanrong, Lu Cheng, Xia Qingyou, et al. Optimization and Improvement of Rapid Amplification of cDNA Ends (RACE) [J]. Life Science Research. 2003,7(3):189-197; [3] Luo Cong, He Xinhua, Chen Hu , et al. A RACE method for efficiently obtaining the 5′ end of a gene [J]. Acta Physiology Plant. 2011, 47(4): 409-414) have improved and optimized each step of RACE (such as selecting different reverse recording enzyme, increasing the reverse transcription temperature, adopting tailing replacement method, adopting hot-start PCR, designing different amplification primers, etc.), but the effect usually needs to be judged after the last two rounds of PCR amplification. For those that affect the experimental results The search for the main effect factor and optimal reaction conditions is inefficient, time-consuming and labor-intensive

Method used

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  • Method for promoting gene cloning efficiency
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  • Method for promoting gene cloning efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1) Extract the total RNA from the liver and head kidney of healthy red-spotted grouper, and test its quality by 1% agarose gel electrophoresis. The buffer system is 0.5×TBE. The results are as follows figure 1 As shown, the 5S, 18S and 28S bands are obvious, indicating that the quality of the extracted RNA is good.

[0029] 2) The first-strand cDNA was synthesized by reverse transcription according to the instructions of the PrimeScriptII 1st Strand cDNA Synthesis kit from TaKaRa.

[0030] 3) Use primers F43 and R167 to detect the product of step 2. The 25 μL system composition of the PCR reaction is: 10×Buffer (containing Mg 2+ ) 2.5 μL, dNTP (dATP, dTTP, dGTP, dCTP each 2.5 mM) 0.5 μL, primers F47 (10 μM) and R167 (10 μM) each 0.5 μL, Taq enzyme (2.5 U / μL) 0.5 μL, template (step 3 The product was diluted 100 times) to 5 μL, and made up to 25 μL with double distilled water; the reaction program was: 95°C for 5 min→(94°C for 30 s, 52°C for 30 s, 72°C for 30 s)×30→72°C ...

Embodiment 2

[0033] 1) Use RNase to degrade the RNA in the reverse transcription product derived from liver tissue.

[0034] 2) Purify cDNA by alcohol precipitation.

[0035] 3) Use primers F43 and R167 to detect the purified product in step 2. The reaction procedure and system composition are the same as step 4 in Example 1. Test results such as image 3As shown, the results show that there is a brighter positive band, which proves that the purification step has been successfully completed.

Embodiment 3

[0037] 1) Using the first-strand cDNA of red-spotted grouper liver as a template, TdT terminal transferase and dATP were used for homopolymer tailing.

[0038] 2) Choose five annealing temperatures of 50°C, 52°C, 54°C, 56°C, and 58°C for the first round of RACE 5' PCR amplification, and the amplification primers are specific primers R167 (5'-CTGAGCCGTCCTGGCACGCAATG-3'), Linker primer RA (5'-AAGCAGTGGTATCAACGCAGAGT-3') and linker primer oligo-dT-RA with oligo-dT, the sequence is 5'-AAGCAGTGGTATCAACGCAGAGTAC(T) 30 VN-3'. After the product was detected by using primers F43 and R167 with the reaction conditions of step 4 of Example 1, it was analyzed by 1.5% agarose gel electrophoresis, and the results were as follows: Figure 4 As shown, the detection bands of amplified products at 50°C and 58°C are the weakest, while the brightness of other temperature gradients are relatively strong, indicating that temperature is one of the important factors affecting the reaction results. I...

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Abstract

The invention discloses a method for promoting gene cloning efficiency, and relates to a gene cloning method. The method comprises the following steps of: designing one pair of specific primers at an intermediate fragment of an object gene; carrying out specific PCR (polymerase chain reaction) amplification on templates of different sources in an RACE (Rapid Amplification of cDNA Ends) process; subjecting an amplification product to the electrophoretic analysis of 1.5% agarose, detecting whether a positive band of the product exists or not and the brightness of the positive band, so as to determine the level of the copy number of the object gene in an RACE to-be-amplified template, and determining the efficiency of each enzymatic reaction step in the RACE process to obtain the most proper template used for 5' RACE amplification, so that the purpose of improving the amplification mission success rate of a target gene is achieved. By using the method, the most proper template for the RACE amplification and a condition are efficiently selected by quickly and sensitively detecting the effective copy numbers of the object genes in the templates of the different sources.

Description

technical field [0001] The invention relates to a gene cloning method, in particular to a method for improving gene cloning efficiency. Background technique [0002] RACE technology has gradually replaced the cDNA library screening technology and has become the main method for cloning new genes due to its advantages of simplicity, rapidity and low cost. However, in actual operation, some difficulties are often encountered, especially 5’-RACE, which is slightly complicated in steps and may encounter many problems. With the increasing improvement of RACE technology, various commercial RACE technology products have been launched. However, there are still many problems in the actual operation of RACE reaction experiments, and it is necessary to repeatedly explore the RACE reaction conditions, especially 5'-RACE. For example, for the classic 5'-RACE, it mainly includes reverse transcription, homopolymer tailing, synthesis of the second strand of cDNA and subsequent nested (neste...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 施晓峰丁少雄王军苏永全
Owner XIAMEN UNIV
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