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Genotyping on ALDH2: comparison of four different technologies.

Zhang L, Zhao J, Cui G, Wang H, Wang DW - PLoS ONE (2015)

Bottom Line: The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing.In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Departments of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Rep. of China.

ABSTRACT

Objectives: This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and methods: We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results: All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions: All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.

No MeSH data available.


Related in: MedlinePlus

The principle and rs671 genotyping results of AS-PCR.A. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct (C-G; T-A). Vice versa, the efficient amplification is low due to the mismatch between the primer and the target sequence, resulting in larger Ct. B. Samples could be distinctly detected upon different Ct value of the two tubes. A genotyping result of GG was shown that the Ct value of the tube containing C allele-specific primer was smaller than the other one comtaining G allele-specific primer.
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pone.0122745.g003: The principle and rs671 genotyping results of AS-PCR.A. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct (C-G; T-A). Vice versa, the efficient amplification is low due to the mismatch between the primer and the target sequence, resulting in larger Ct. B. Samples could be distinctly detected upon different Ct value of the two tubes. A genotyping result of GG was shown that the Ct value of the tube containing C allele-specific primer was smaller than the other one comtaining G allele-specific primer.

Mentions: We also tested the performance of AS-PCR for genotyping ALDH2 (Fig 3). Different from above-mentioned methods, each sample was detected in at least two vials with different reverse allele-specific primers by AS-PCR. PCR reactions of the two vials had no difference except for the allele-specific primer, one matching to allele G and the other to A of rs671. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct. In contrast, the efficiency of amplification was low with mismatch between the primer and the target sequence, resulting in larger Ct (Fig 3A). Samples could be distinctly detected upon different Ct value of the two vials (Fig 3B). To increase the allele-specificity, we introduced an additional primer mismatch at the third nucleotide from the 3' end of the primers.


Genotyping on ALDH2: comparison of four different technologies.

Zhang L, Zhao J, Cui G, Wang H, Wang DW - PLoS ONE (2015)

The principle and rs671 genotyping results of AS-PCR.A. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct (C-G; T-A). Vice versa, the efficient amplification is low due to the mismatch between the primer and the target sequence, resulting in larger Ct. B. Samples could be distinctly detected upon different Ct value of the two tubes. A genotyping result of GG was shown that the Ct value of the tube containing C allele-specific primer was smaller than the other one comtaining G allele-specific primer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372573&req=5

pone.0122745.g003: The principle and rs671 genotyping results of AS-PCR.A. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct (C-G; T-A). Vice versa, the efficient amplification is low due to the mismatch between the primer and the target sequence, resulting in larger Ct. B. Samples could be distinctly detected upon different Ct value of the two tubes. A genotyping result of GG was shown that the Ct value of the tube containing C allele-specific primer was smaller than the other one comtaining G allele-specific primer.
Mentions: We also tested the performance of AS-PCR for genotyping ALDH2 (Fig 3). Different from above-mentioned methods, each sample was detected in at least two vials with different reverse allele-specific primers by AS-PCR. PCR reactions of the two vials had no difference except for the allele-specific primer, one matching to allele G and the other to A of rs671. The binding efficiency of allele-specific primer to target sequence was high when they matched well at the 3′ end of the primer, resulting in efficient amplification with smaller Ct. In contrast, the efficiency of amplification was low with mismatch between the primer and the target sequence, resulting in larger Ct (Fig 3A). Samples could be distinctly detected upon different Ct value of the two vials (Fig 3B). To increase the allele-specificity, we introduced an additional primer mismatch at the third nucleotide from the 3' end of the primers.

Bottom Line: The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing.In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.

View Article: PubMed Central - PubMed

Affiliation: Departments of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Rep. of China.

ABSTRACT

Objectives: This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and methods: We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results: All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions: All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.

No MeSH data available.


Related in: MedlinePlus