Genomic DNA was extracted from EDTA-containing peripheral blood samples (commercial kit FlexiGene® DNA kit, Qiagen, Germany). Genotyping of SNPs rs2583988 (C > T), rs356219 (A > G), rs2736990 (T > C), and rs11931074 (G > T) in SNCA gene was performed using real-time TaqMan® polymerase chain reaction assay according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, United States). Four samples were lost due to poor quality of DNA. 104 PD patients and 98 controls were successfully genotyped.
Taqman polymerase chain reaction assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan® polymerase chain reaction (PCR) assay is a molecular biology technique used to amplify and detect specific DNA sequences. It employs a fluorescent probe to monitor the amplification of the target DNA in real-time. The assay utilizes a thermostable DNA polymerase enzyme to replicate the target DNA, enabling the quantification of the initial DNA template.
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4 protocols using taqman polymerase chain reaction assay
1
SNCA Gene Polymorphisms in Parkinson's Disease
2
Validating Genetic Associations with Sex Ratio
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Genetic associations with sex ratio were further examined using an independent Korean population to validate genetic associations revealed in the current GWAS. This included 935 subjects recruited from routine health checkups at Hallym University Hospital. 18, 19 Genomic DNA was extracted from their peripheral blood cells using QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). Nucleotide variants were genotyped using the TaqMan polymerase chain reaction assay (Applied Biosystems, Foster City, CA, USA). All reactions were carried out following the manufacturer's protocol, and the products resulted from the reactions were analyzed using ABI PRISM 7900HT (Applied Biosystems). Genetic associations were determined by the Fisher's exact test under the assumption of an additive contribution of minor allele. Multiple testing by Bonferroni correction was also conducted for the association analysis.
3
STI Screening and Pregnancy History
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Participants' self-collected vaginal swabs (Swube applicator; Becton Dickinson Microbiology Systems, Sparks, MD) were evaluated for Chlamydia trachomatis and Neisseria gonorrhoeae using BD ProbeTec ET GC/CT-Amplified DNA assays (Becton, Dickinson and Company)21 and for Trichomonas vaginalis using TaqMan polymerase chain reaction assay (Thermo Fisher Scientific, Waltham, MA). For these analyses, we created a composite variable, whereby women who tested positive for one or more STIs were coded as “1” and those who tested negative for all 3 were coded as “0.”
In addition, we included 2 questions related to pregnancy history as proxies for occurrence of unprotected sex: ever having been pregnant (0 = No; 1 = Yes) and ever having an abortion or miscarriage (0 = No; 1 = Yes).
In addition, we included 2 questions related to pregnancy history as proxies for occurrence of unprotected sex: ever having been pregnant (0 = No; 1 = Yes) and ever having an abortion or miscarriage (0 = No; 1 = Yes).
4
STI Screening and Pregnancy History Protocol
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