DES-Testbed, Freie Universität Research Plant,Polyclonal,Real-time Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism

Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism


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Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism associated with Gilbert’s syndrome.

BACKGROUND

Gilbert’s syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians.

The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay.

METHODS

Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes- (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis.

RESULTS

All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes.

CONCLUSIONS

We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.

Systematic analysis of mitochondrial genes associated with hearing loss in the Japanese population: dHPLC reveals a new candidate mutation.

BACKGROUND

Variants of mitochondrial DNA (mtDNA) have been evaluated for their association with hearing loss. Although ethnic background affects the spectrum of mtDNA variants, systematic mutational analysis of mtDNA in Japanese patients with hearing loss has not been reported.

METHODS

Using denaturing high-performance liquid chromatography combined with direct sequencing and cloning-sequencing, Japanese patients with prelingual (N = 54) or postlingual (N = 80) sensorineural hearing loss not having pathogenic mutations of m.1555A>> G and m.3243A>> G nor GJB2 were subjected to mutational analysis of mtDNA genes (12S rRNA, tRNALeu(UUR), tRNASer(UCN), tRNALys, tRNAHis, tRNASer(AGY), and tRNAGlu).

RESULTS

We discovered 15 variants in 12S rRNA and one homoplasmic m.7501A>> G variant in tRNASer(UCN); no variants were detected in the other genes. Two criteria, namely the low frequency in the controls and the high conservation among animals, selected the m.904C>> T and the m.1105T>> C variants in 12S rRNA as candidate pathogenic mutations. Alterations in the secondary structures of the two variant transcripts as well as that of m.7501A>> G in tRNASer(UCN) were predicted.

CONCLUSIONS

The m.904C>> T variant was found to be a new candidate mutation associated with hearing loss. The m.1105T>> C variant is unlikely to be pathogenic. The pathogenicity of the homoplasmic m.7501T>> A variant awaits further study.

Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC).

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based denaturing high-performance liquid chromatography (DHPLC).

An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms.

The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications.

A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.

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des-testbed

Analysis of human glutathione S-transferase alpha 1 (hGSTA1) gene promoter polymorphism using denaturing high performance liquid chromatography (DHPLC).

OBJECTIVE

The GST enzyme, encoded by hGSTA1 gene, catalyses the GSH dependant detoxification of a variety of carcinogenic metabolites and alkylating chemotherapeutic agents. Two genetic variants of hGSTA1, namely hGSTA1*A and hGSTA1*B, are characterized by three linked SNPs, of which -52 G>A variation being solely responsible for the differential promoter activity of hGSTA1.

Individuals homozygous for hGSTA1*B have low hepatic expression of hGSTA1. Given the time and labor consuming PCR-RFLP method and the direct prediction of -52 G>A variation, we opted to establish a high throughput DHPLC procedure for the characterization of hGSTA1 variants.

METHODS

117 DNA samples from South India were included in the study. Control samples were generated for DHPLC using conventional PCR-RFLP technique. Heteroduplexes were produced by in vitro mixing of control DNA samples (hGSTA1*A) to all the samples which are subsequently subjected to DHPLC analysis. The samples were analyzed for the presence of heteroduplexes from the chromatographic profiles.

CONCLUSIONS

From the total of 117 samples, 43.5% are homozygous for hGSTA1*A allele, 13% are homozygous for hGSTA1*B allele and 43.5% are hGSTA1*A/B heterozygotes. This is, to our knowledge, the first report on the use of DHPLC for the evaluation of hGSTA1 gene promoter polymorphism.

Identification of mutations in the lipoprotein lipase (LPL) and apolipoprotein C-II (APOC2) genes using denaturing high performance liquid chromatography (DHPLC).

BACKGROUND

Endothelial lipoprotein lipase (LPL) hydrolyzes triglycerides of chylomicrons and very low density lipoproteins, releasing free fatty acids for local and systemic use. Mutations in the LPL gene or its cofactor APOC2 may result in a decrease or complete loss of enzyme function and subsequently to type I hyperlipoproteinemia.

METHODS

We used PCR to amplify all exons and the promoter region of LPL and APOC2. Nine blinded DNA samples with known LPL mutations were used as positive controls. In addition, nine patients from our lipid clinic and twelve healthy subjects were analyzed. DNA was screened for sequence variants by denaturing HPLC (DHPLC) followed by direct sequencing of PCR fragments showing distinct elution profiles.

RESULTS

All LPL sequence variants in the positive controls (D9N, V69L, delAACTG386, I225T, N291S, and S447X) were correctly identified. In the remaining patients, additional variants were detected in LPL and APOC2. These variants were also present in healthy subjects, indicating that they constituted silent variation with no relevant effect on plasma triglycerides, at least in the heterozygous state.

CONCLUSIONS

A semi-automated DHPLC screening method was developed for the detection of sequence variants in the LPL and APOC2 genes. Our results demonstrate that the method was robust and sensitive.

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