Rapid Diagnosis Of 21 Trisomy Syndrome By SYBR Green I Homologous Genes Quantitative Polymerase Chain Reaction (HGQ-PCR)

Objectives: Establishing a practical and efficient method used in clinical and prenatal diagnosis . Methods 1. Establishing methods We designed a method named the SYBR Green I staining homologous Genes quantitative polymerase chain reaction (HGQ-PCR),It can directly identify the additional copy of chromosome 21.At first, using one pair of primers to simultaneously amplify different fragments of two highly homologous genes of the human liver-type phosphofructokinase located on chromosome 21(PFKL-CH21) in exon 8 and the human muscle-type phosphofructokinase located on chromosome 1(PFKM-CH1) in exon 9 for self-detection determination.Then, staining the PCR products of these homologous genes after low number cycles.At last., comparing the fluorescence intensity of the bands and analysing on Bio-Rad Gel Doc 2000. 2. Detecting DS:DNA was isolated from the blood samples of 26 DS cases and 20 normal individuals who were diagnosed by karyotype analysis before.All samples were detected by HGQ-PCR .3. Comparing with the others methods : We compared SYBR Green I with Ethidium bromide and silver-staining to observe the sensitivity. Results 1. On analysis of the DS cases and normal individuals by SYBR Green I HGQ-PCR assays, the fluorescence intensity relative ratio of PFKL-CH21/PFKM-CH1 PCR products were 1.61±0.18(mean±SD) and 1.01±0.06(mean±SD) , respectivly．The difference between the two groups was highly significant(P<0.01).The results were consistent with expections.It comfired that using our method to dognose DS was the same accurate as by themeans of karyotype analysis, furthermore, it is faster than the later. 2．SYBR Green I stain is as sensitive as silver-staining but more simple and rapid than the later. Comparing with Ethidium bromide, it is more sensitive,accurate and safe. Conclusion 1. SYBR Green I HGQ-PCR is a practical approach with rapid and simple characters,which can be used for the clinical and prenatal diagnosis of DS.2. DS was diagnosed accurately by SYBR Green I HGQ-PCR assays.The results were consistent with karyotype analysis.But the former is more rapid, simple and economic than the later.3. SYBR Green I is a sensitive DNA stain, It can be used to identify low quantity DNA,including the PCR products after low number cycles．4.Using one pair of primers to amplify two highly homologous genes simultaneously can increase the accuracy of Q-PCR.