Analytical Methods Of Gene Gene Copy Number Variations Based On Multiplex Competitive Pcr Using Universal Fluorescent Primers

As a complex genetic variation in the human genome, copy number variation has great significance in disease research. At present, the CNV detection technology has been largely commercialized, therefore which has a high cost and experimental equipment requirements. A multiplex Competitive PCR technology with a universal fluorescent primer was established. By regulating primer annealing temperature, PCR reaction was conducted in two phases. In the first10cycles, the chimeric forward primers are used to amplify the DNA template. As the reaction proceeds, Universal fluorescent primers complete the entire amplification reaction. PCR products were separated and detected by ABI sequencing instrument for the peak. Standardized peaks of the test sample were compared with control samples to detect the copy number of the test sample. Universal primers can reduce the cost of the experiment, synchronize amplification of all loci and greatly reduces the complexity of the multiplex PCR, making it easy to achieve the quantitative detection of CNV. The paper also raises a new program, which is used to prepare internal control. The contents of this program are as follows:The2bp base insertions or deletions were introduced to the target segment by PCR; PCR products were cloned into a T vector; Verified by sequencing, cloning plasmid was extracted; Be digested, plasmid can be used as internal control; Internal control for the first time preparation is slightly complicated, but Control within a preparation, can be long-term use. The program is simple, economical and easy to implement.The three test sites, which have been test by this technology, have been designed in transgenic section of three mice (ZJY-1、ZJY-2、ZJY-3). The results showed that ZJY-1, ZJY-2and ZJY-3target segments is followed by four copies, three copies and five copies. The test results are consistent with the results of real-time of qPCR. The experimental results also showed that the technology has a high sensitivity that single-copy changes can be test within the five copies. The technology with a certain test flux can meet the general test requirements. Subsequently, some factors, which affect the experimental accuracy,have been studied. The main factors are as follows:the structure morphology of internal control, the2bp sequence differences, PCR volume, the relative amount between test sample and control, universal primers to add or not, functional relationship between the fluorescence signal intensity and amount of PCR product. The experimental results are as follows:When have similar spatial structure, Internal control and test samples have similar amplification efficiency; Only with the similar initial amount of template or high levels of the test sample, experiment has a good test effect; the PCR reaction volume should be less than5ul, otherwise the experimental results will be biased. Experimental data show that fluorescence signal intensity can be approximated as proportional to the amount of PCR product. The intensity of the fluorescence signal can be directly used for analysis of the CNV, without the need for correction through the formula. Other factors had no significant effect on the experimental results.