Probe Melting Curve Analysis For The Detection Of Chromosomal Microdeletions And Microduplications

Chromosomal microdeletions and microduplications are related to many diseases.For example,the 22q11.2 microdeletion syndrome,22q11.2 microduplication syndrome,7q11.23 microdeletion syndrome and 7q11.23 microduplication syndrome.They are all the common chromosomal disorders.Their clinical phenotype is complex and diverse which endangers normal development of multiple organs and the prognosis is poor.So that early screening and diagnosis are extremely important.Real-time fluorescent quantitative PCR technology as an important tool,has been widely used in point mutations,copy number variation,and so on.It has many advantages,such as short detection time,high sensitivity,high degree of automation,specificity and closed tube pollution-free operation.In this study,real-time fluorescence quantitative PCR technique were used to detect the four common chromosomal microdeletions and microduplications diseases which based on the DNA sequence similarity.Fluorescent probe melting curve analysis use a fluorescent probe which can combine with the target sequence and reference sequence,after a melting it will produce specific Tm of the target sequence and reference sequence.According to the Ratio value we can calculate the copy number of the target sequences.With only a pair of primers,we can realize the simultaneous amplification of target sequences and similar sequences,so the consistency of PCR amplification efficiency can be guaranteed.Finally,by normalizing the Ratio values obtained from the three detection fragments,the relative copy number can be obtained.Two genes(DGCR5 and UFD1L)in the deleted area of 22q11.2 DS were selected and total of 8 test sites were selected to be detected and three detection sites were selected after screening,two located in DGCR5 and one is located in UFD1L.The reference sequences are located in chr17,chr18 and chr22 respectively.Results show that 22q11.2 DS,22q11.2 microduplication syndrome detection system with minimum detectable limit is 5 ng pere reaction.Data of the two same experiments were consistent with P>0.05(Student’s test),indicating there is no statistical difference.In addition,the three genotype plasmid standards can be divided into different regions and can simulate the actual samples.805 blood samples were detected and the SD value is 0.0610,the CV value is 0.0629 and the threshold value is 0.9699±0.1220(mean±2SD).We also detected 196 saliva samples with the SD is 0.0691,the CV value is 0.0712,and the threshold value is 0.9700±0.1380(mean±2SD).The threshold value of saliva samples is very close to the blood samples but the stability of the saliva samples is slightly worse and it is related to there poor quality.In the four clinical specimens,the relative copy number of a heterozygous deleted specimen is 0.4842,and two wild-type specimens are 0.9325 and 1.0066 respectively,and a duplicated sample is 1,9709.The results were consistent with the clinical diagnosis.For 7q11.23 microdeletion syndrome and 7q11.23 microduplication syndrome,BAZIB,GTF2IRD1 and the spacer between WBSCR28 and ELNwere selected and total of 8 test sites to be designed and three detection sites were selected after screening,two sites located in GTF21RD1,1 site is located in the interval district of WBSCR28 and ELN,reference sequences are located on the chr7:RABGEF1 and chr7:LOC100420647.7q11.23 microdeletion syndrome and 7q11.23 microduplication syndrome detection system can detect as least 1.25 ng DNA;Data of the two same experiments were consistent with P>0.05;three genotype plasmid standards can be well distinguished.A total of 839 blood samples were detected with the SD is 0.0417,CV is 0.0413,and the threshold value is 1.0097±0.0965(mean±2SD).We also detected 201 saliva samples and the CV value is 0.0476,the SD is 0.0466,and the threshold value is 0.9798±0.0932(mean±2SD).The threshold value of saliva samples is very close to the blood samples.In the four clinical specimens,the relative copy number of a heterozygous deleted specimen is 0.5820,and two wild-type specimens are 0.9779 and 1.0754 respectively,and a duplicated sample is 1.5920.The results were consistent with the clinical diagnosis.Current clinical diagnostic methods are often time-consuming and high cost.By contrast,the real-time fluorescent PCR melting curve analysis which based on DNA similarity sequence principle has many advantages,such as simple,rapid,sensitive,high flux,automation,closed tube operation no pollution,low cost.It will be a suitable method for prenatal diagnosis and early screening.