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Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array.

Koo SH, Ong TC, Chong KT, Lee CG, Chew FT, Lee EJ - Biol Proced Online (2007)

Bottom Line: Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management.The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping.It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

View Article: PubMed Central - PubMed

Affiliation: Singapore Department of Pharmacology, National University of Singapore.

ABSTRACT
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

No MeSH data available.


Related in: MedlinePlus

Fig. 2Representative allelic ratio scatterplots (A) rs1045642 and (B) rs562.
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f2: Fig. 2Representative allelic ratio scatterplots (A) rs1045642 and (B) rs562.

Mentions: Tables 6 and 7 show the raw MFI values of each individual sample (assayed in duplicates) and a non-template background control. The results between the duplicates for all readouts were clearly consistent. The genotyping assay for the 22 SNPs was highly specific and robust; the signal-to-noise ratio (specific MFI/background) ranged from >10 to >900 and the MFI units ranged from approximately 300 to 25,000. Threshold values were empirically determined for each individual SNP. The mutant allelic ratio ranged from <0.01-0.28 for wild-type calls, 0.19–0.78 for heterozygous calls, and 0.89–1.00 for mutant calls. While the heterozygotes exhibited almost equal MFI signals for both alleles for the majority of the SNPs, the MFI signal of one allele was higher than the other for a few SNPs. This could possibly be explained by the reasoning that one allele of a SNP amplified disproportionately and obscured the presence of the other allele. Nonetheless, with prior validation using known control samples, the various genotypes are clearly distinguishable. Representative allelic ratio scatterplots are shown for two of the SNPs (Fig. 2).


Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array.

Koo SH, Ong TC, Chong KT, Lee CG, Chew FT, Lee EJ - Biol Proced Online (2007)

Fig. 2Representative allelic ratio scatterplots (A) rs1045642 and (B) rs562.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211573&req=5

f2: Fig. 2Representative allelic ratio scatterplots (A) rs1045642 and (B) rs562.
Mentions: Tables 6 and 7 show the raw MFI values of each individual sample (assayed in duplicates) and a non-template background control. The results between the duplicates for all readouts were clearly consistent. The genotyping assay for the 22 SNPs was highly specific and robust; the signal-to-noise ratio (specific MFI/background) ranged from >10 to >900 and the MFI units ranged from approximately 300 to 25,000. Threshold values were empirically determined for each individual SNP. The mutant allelic ratio ranged from <0.01-0.28 for wild-type calls, 0.19–0.78 for heterozygous calls, and 0.89–1.00 for mutant calls. While the heterozygotes exhibited almost equal MFI signals for both alleles for the majority of the SNPs, the MFI signal of one allele was higher than the other for a few SNPs. This could possibly be explained by the reasoning that one allele of a SNP amplified disproportionately and obscured the presence of the other allele. Nonetheless, with prior validation using known control samples, the various genotypes are clearly distinguishable. Representative allelic ratio scatterplots are shown for two of the SNPs (Fig. 2).

Bottom Line: Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management.The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping.It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

View Article: PubMed Central - PubMed

Affiliation: Singapore Department of Pharmacology, National University of Singapore.

ABSTRACT
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

No MeSH data available.


Related in: MedlinePlus