Using Two Colour Real Time Fluorescence Quantitative PCR And Fluorescence In Situ Hybridizationto Detect Down Syndrome

Backgrounds and Purposes: Down Syndrome is one of the most common normal chromosome anomaly in live birth . Classically examinations are lengthy procedures relying on karyotypic analysis of cultured peripheral blood lymphocyte or amniocytes or chorionic mesenchyme.Recent studies have shown that real-time PCR can also be used to detect more subtle increments in gene dosage, such as hetero- or homozygosity of the RhD locus and protooncogene amplification in breast cancer patients. Here we develope a two colour real-time fluorescence quantitative PCR assay for the detection of Down Syndrome that is readily amenable to automation and high-throughput diagnosis.At the same time ,we also use fluorescence in situ hybridization to detect Down Syndrome and to demonstrate the PCR results.Methods:The DNAs of the peripheral blood lymphocyte or amniocytes were extracted by phenol trichlormethane method . TaqMan probe of the Down Syndrome critical region gene 1(DSCR1)and control gene glyceraldehydes 3-phosphate dehydrogenase(GAPD) were labeled by FAM and HEX fluorescence dye separately.DSCR1 and GAPD were coamplified simultaneously in a multiple reaction Q-PCR . CT_DSCR1 and CTgapd of each specimen were recorded by Q-PCR instrument,each specimen's ACT are CT_DSCR1-CT_GAPD which are used to determine whether it was patient,andused to statistics. Each specimen was detected three times ,and the DNA were diluted by 10-fold.The three ACTs were averaged. The amplification efficiency were determined by DNA dilution .We used DIG labeled DSCRJ gene probes and fluorescence labeled 21 chromosome q22 probes to hybridize DSCR1 gene located in 21 chromosome.Results:We detected 27 normal cases and 25 Down Syndrome patients'peripheral blood lymphosate DNA,and 15 normal and 2 Down Syndrome amniocytes DNA. The ACT of normal cases had mean ratios of —0.34 ±0.21,the ACT of Down Syndrome patients had mean ratios of— 1.332 + 0.368.There have significant statistic difference in ACT.At last we could conclude that the patients DSCR1 dosage are 1.79 + 0.15 of normal groupe .In fluorescence in situ hybridization using DIG labeled and fluorescence labeled probe,we found there were two hybridization loci in normal cases chromosome or interphase nucleus,and there were three hybridization loci found in patients specimens chromosome and interphase nucleus .These two methods gave the same results.Conclusion :The real-time PCR with two colour TaqMan probes for two genes reacted in the same tube can be correctly used to detect Down Syndrome . Two colour fluorescence real-time PCR assay in which two gene(DSCRl and GAPD) are coamplified to decrease the difference between these gene and gets the almost coincidence efficiency.lt is possible to determine the relative ratio(ACx) of these genes .The approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome . It is also applicable to aneuploidies other than trisomy 21 .The fluorescence in situ hybridization is also a rapid and accurate method to detect Down syndrome if we can use it to detect the uncultured amniocytes interphase nucleus.