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Application of SYBR Green I Real-time PCR in ABO Genotyping

Author: LiGuiNing
Tutor: HuLiHua
School: Huazhong University of Science and Technology
Course: Clinical Laboratory Diagnostics
Keywords: ABO blood group PCR Gene polymorphism Real-time PCR SYBR Green I Artificially mismatched base
CLC: R440
Type: Master's thesis
Year: 2011
Downloads: 13
Quote: 0
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Abstract


ABO genotyping method for the purpose of establishing a common set of PCR , using this method to filter out common ABO genotype . 66 volunteers were blood specimens, collected blood type is positive and negative stereotypes consistent ABO gene polymorphism loci 261,467,802,803,1061 genotypes were identified using conventional PCR and sequenced to verify the specificity . Results ordinary PCR screened eight kinds the common ABO diploid genotype A101 / O01 , 02 in 7 cases , A102 / O01, 02 type 8 cases A101/A102 type 3 cases , three cases B101/B101 B101 / O01 02 type 14 cases , O01 , 02 / O01 , 02 of 18 cases , the A101/B101 type 3 cases , A102/B101 10 cases , consistent with the phenotypic identification results . The conclusion we established the ordinary PCR genotyping method is reliable , the next step SYBR Green I real-time PCR method can be used as reference and evaluation criteria . Objective To investigate the SYBR Green I real-time PCR in blood typing . Method we created in the first part of ordinary PCR typing method to filter out the best primers ABO phenotypes and genotypes screened design the mismatched primers suitable for SYBR Green I real-time PCR , melting curve of 261,467 803 points were detected, and compared with normal PCR . Best wrong results real-time PCR with primers for 261nor - 260 ( A → T ) , 261mut - 260 ( C → T ) , 467mut - 466 ( T → G) 803nor -804 ( T → G) 803mut -804 ( T → G), using the melting curve of 261,467,803 locus genotyped , without significant interference . SYBR Green I real-time PCR is fast, accurate , sensitivity up 12.5ng . Conclusion PCR is simple , but by the template the quality and reaction conditions affect the greater use of SYBR Green I real-time PCR melting curve analysis can quickly and accurately identify the genotype of blood group polymorphisms (SNPs) .

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