A method for obtaining myc gene in human tissue by two-step overlapping PCR method

Abstract

The invention belongs to the field of biotechnology, and relates to a method for obtaining MYC gene in human tissue by using a two-step overlapping PCR method. The method includes: (1) TES water bath dewaxing; (2) extracting DNA from human tissue samples; (3) finding and selecting 7 MYC gene sequences with the same length; (4) designing 16 specific primers for MYC genes (5) two-step overlapping PCR amplification; (6) purifying DNA fragments; (7) measuring DNA concentration; (8) gene detection. Utilizing the present invention, the accuracy is high, the efficiency is high, the dewaxing effect is better, the damage to the environment and the human body is smaller, the operation is simple, the cost is low, and the DNA fragment of the MYC target gene of 5279bp different sequences is amplified, and the purity of the target gene And the concentration is high, and the fragment of the target gene is verified by the sequencing method, which provides convenience for biomedical research.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for obtaining MYC gene in human tissue by using a two-step overlapping PCR method. Background technique [0002] Formalin-fixed paraffin-embedded technique is the most widely used in clinicopathological diagnosis. In the formalin-fixed paraffin embedding technique, formalin, as a fixative, can reduce the change of tissue morphology within a certain period of time, and provide reliable information for subsequent immunohistochemistry, histological analysis and genetic diagnosis. Assure. [0003] The existing formalin-fixed paraffin embedding technology still has the following defects: First, the DNA extracted from the specimen has a low concentration and short fragments. As time goes by, the quality of the specimen gradually decreases, making it difficult to obtain a complete gene It becomes quite difficult, and the fragmentation of DNA will lead to the lack of integrity of th...

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Problems solved by technology

[0003] The existing formalin-fixed paraffin embedding technology still has the following defects: First, the DNA extracted from the specimen has a low concentration and short fragments. As time goes by, the quality of the specimen gradually decreases, making it difficult to obtain a complete gene It becomes quite difficult, and the fragmentation of DNA will lead to the lack of integrity of the target gene, making the test results unreliable and data splicing more difficult, making it impossible to analyze the haplotype information of the full-length MYC gene, causing serious damage to clinical testing and medical science research. It is a big obstacle; secondly, the xylene dewaxing method is often used in the existing paraffin-embedded tissue dewaxing method, but xylene is easy to pollute the environment, is harmful to the human body, and the extraction process is long; thirdly, the current method of obtaining the target DNA fragment The most common methods include PCR, enzyme digestion, gene chips, hybrid capture, etc., but the obtained fragments have problems such as easy breakage, low purity and concentration, more impurities, and short length. At the same time, they are generally used in formalin-fixed paraffin The next-generation sequencing method for DNA detection embedded in human tissue also has problems such as short read length, too large sequencing depth, and insensitivity to high GC regions, repeated regions, and base-modified regions, which makes the amount of information spliced ​​​​large and cannot be combined. MYC gene testing, which increases the difficulty of analyzing long-segment DNA results

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