Method for preparing standard reference for qualitative, quantitative DNA

Abstract

This invention discloses a simple and low-cost method for preparing qualitative and quantitative DNA marker. The DNA marker is prepared by PCR. In the PCR, the template is an artificially synthesized DNA fragment containing repeated DNA sequence, while the primers are the same or complementary with the repeated sequence. The template and primer are placed in a single reaction tube to perform PCR and obtain the needed DNA marker. The template contains 15-30 bp repeated DNA fragments, the repeating number of the repeated sequence is not more than 50, and the alkali number between two repeated sequences is integer times of 20 bp.

Description

Technical field: [0001] The invention relates to a genetic engineering, in particular to a method for preparing a qualitative and quantitative deoxyribonucleic acid standard reference substance. Background technique: [0002] The deoxyribonucleic acid standard reference substance is composed of a series of known deoxyribonucleic acid fragments with different numbers of bases. There are two types of preparation methods. The first type of method is enzymatic digestion, which has defects in natural genes and phage DNA methods. The reason is that the fragments are not evenly distributed, and there is often a large gap between some fragments, which makes it impossible to scale the region more accurately. However, the preparation steps of the artificially constructed vector by enzyme digestion are cumbersome, and in The operation process of conversion, extraction and enzyme digestion requires high technical difficulty and is not suitable for industrial production. The second type...

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Problems solved by technology

[0002] The deoxyribonucleic acid standard reference substance is composed of a series of known deoxyribonucleic acid fragments with different numbers of bases. There are two types of preparation methods. The first type of method is enzymatic digestion, which has defects in natural genes and phage DNA methods. The reason is that the fragments are not evenly distributed, and there is often a large gap between some fragments, which makes it impossible to scale the region more accurately. However, the preparation steps of the artificially constructed vector by enzyme digestion are cumbersome, and in The operation process of conversion, extraction and enzyme digestion requires high technical difficulty and is not suitable for industrial production

The second type of method is the polymerase chain reaction (PCR) method, by designing a series of PCR products that can amplify the length of DNA with different base numbers to obtain a series of DNA fragments of different lengths, and then purifying and mixing the DNA fragments of different lengths to prepare This kind of PCR amplification prepares gradient deoxyribonucleic acid standard reference substance. The preparation steps are relatively simple, which overcomes the disadvantages of many enzyme digestion steps and high difficulty of preparation process. However, the disadvantage of this method is that due to multiple Different PCR primers are used to amplify DNA bands in different reaction tubes, which requires a lot of equipment; because different DNA bands come from different reaction tubes, there is no definite proportional relationship between the DNA fragments in each tube, giving The deployment of different DNA bands in the later stage causes difficulties and lacks quantitative functions; in addition, when this method requires gel purification, it increases the chance of single-strand breaks in double-stranded DNA molecules. Due to the use of many reaction tubes and reagents, increased manufacturing costs