Genomic DNA Extraction and Genotyping

We had extracted Genomic DNA from 200 μL of blood collected from each participant using a TIANamp Blood DNA Kit (Tiangen, Beijing city, China) and we followed the specific procedures in the literature (35 (link)–37 (link)). The extracted DNA is placed in a −80°C refrigerator until use. The allele-specific probes were purchased from Applied Biosystems. TaqMan real-time polymerase chain reaction of the samples is performed in 384-well plate with an ABI Q6 instrument (Thermo Fisher Scientific, Waltham, MA, United States) to genotype rs7404339 polymorphism in CDH5 (38 (link), 39 (link)). For quality control, each 384-well plate contained eight samples without DNA but with the same amount of distilled water. Moreover, to ensure the quality and accuracy of the genotyping results, we randomly selected 10% of the samples for a repeat analysis, and the results were 100% concordant.

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Wang Y., Lin K., Zhang L., Lin Y., Yu H., Xu Y., Fu L., Pi L., Li J., Mai H., Wei B., Jiang Z., Che D, & Gu X. (2022). The rs7404339 AA Genotype in CDH5 Contributes to Increased Risks of Kawasaki Disease and Coronary Artery Lesions in a Southern Chinese Child Population. Frontiers in Cardiovascular Medicine, 9, 760982.

Publication 2022

TIANamp Blood DNA Kit allele-specific probes ABI Q6 instrument

Allele Blood Cdh5 Genomic Polymorphism Real time polymerase chain reaction

Corresponding Organization : Guangzhou Women and Children Medical Center

Top 5 similar protocols

Variable analysis

independent variables

Rs7404339 polymorphism in CDH5

dependent variables

Genotyping results

control variables

Amount of blood collected from each participant (200 μL)
TIANamp Blood DNA Kit (Tiangen, Beijing city, China) used for DNA extraction
Specific procedures in the literature (35-37) followed for DNA extraction
Extracted DNA stored in a -80°C refrigerator until use
Allele-specific probes purchased from Applied Biosystems
TaqMan real-time polymerase chain reaction performed in 384-well plate with an ABI Q6 instrument (Thermo Fisher Scientific, Waltham, MA, United States)
Eight samples without DNA but with the same amount of distilled water included in each 384-well plate for quality control
10% of the samples randomly selected for repeat analysis, with 100% concordance

negative controls

Eight samples without DNA but with the same amount of distilled water included in each 384-well plate for quality control

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