A novel multiplex fluorescent competitive PCR for copy number variation detection |
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| Institution: | 1. College of Environmental Science and Engineering, Donghua University, Shanghai, China;2. Obstetrics and Gynecology Hospital, State Key Laboratory of Genetic Engineering at School of Life Sciences, Institute of Reproduction and Development, Fudan University, Shanghai, China;3. Key Laboratory of Reproduction Regulation of NHFPC, Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai, China;4. Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China;5. Department of Orthopedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China;6. Beijing Key Laboratory for Genetic Research of Skeletal Deformity, Beijing, China;7. Medical Research Center of Orthopedics, Chinese Academy of Medical Sciences, Beijing, China;8. Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, China |
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| Abstract: | The copy number variation (CNV) is an important genetic marker in cancer and other diseases. To detect CNVs of specific genetic loci, the multiplex ligation-dependent probe amplification (MLPA) is an appropriate approach, but the experimental optimization and probe synthesis are still great challenges. The multiplex competitive PCR is an alternative method for CNV detection. However, the construction of internal competitive template and establishment of a stable multiplex PCR system are the main limiting factors for this method. Here, we introduce a novel multiplex fluorescent competitive PCR (NMFC-PCR) for detecting CNVs. In this method, the blunt hairpin primers are used to rapidly establish a stable multiplex PCR system due to the reduction of non-specific amplification, and limited cycles' amplification is used to obtain the internal competitive template instead of artificial synthesis. With this method, we tested 21 clinical samples with potential LIM homeobox 1 (LHX1) or T-box 6 (TBX6) deletion. Every three segments located on the LHX1 and TBX6 were selected as the target regions, while two segments located on X-chromosome and five segments located on autosome were selected as the reference regions for detecting CNVs. The results showed that the gender information of 21 samples can be accurately inferred by the copy number ratio (CNR) of X-chromosomal reference region to autosomal reference region (X/A), and 2 samples had one copy of LHX1 and 9 samples had one copy of TBX6. To evaluate the accuracy of NMFC-PCR, 5 random samples with CNV were also detected by array-based comparative genomic hybridization (aCGH), and the results of aCGH were consistent with the NMFC-PCR results. To further assess the performance of NMFC-PCR, 60 normal samples were simultaneously tested. The results showed that the gender results were exactly the same as known information, and CNVs of LHX1 or TBX6 were not found. In conclusion, the method is a cheap, efficient, accurate, and convenient competitive PCR method for CNV detection. |
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