Rapid, Long-Range Molecular Haplotyping of Thiopurine S-Methyltransferase (TPMT*) *3A, *3B, and *3C

Abstract Background: Haplotyping is an important technique in molecular diagnostics because haplotypes are often more predictive for individual phenotypes than are the underlying single-nucleotide polymorphisms (SNPs). Until recently, methods for haplotyping SNPs separated by kilobase distances were laborious and not applicable to high-throughput screening. In the case of thiopurine S-methyltransferase (TPMT*), differentiating among TPMT*3A, *3B, and *3C alleles is sometimes necessary for predictive genotyping. Methods: The genomic region including the two SNPs that define TPMT*3A, *3B, and *3C alleles was amplified by long-range PCR. The resulting PCR product was circularized by ligation and haplotyped by allele-specific amplification PCR followed by product identification with hybridization probes. Results: Critical points were the long-range PCR conditions, including choice of buffer and primers, optimization of the ligation reaction, and selection of primers that allowed for strict allele-specific amplification in the second-round PCR. Different underlying TPMT haplotypes could then be differentiated. Results from the haplotyping method were in full agreement with those from our standard real-time PCR method: TPMT*1/*3A (n = 20); TPMT*1/*3C (n = 4); TPMT*1/*1 (n = 6); and TPMT*3A/*3A (n = 6). One TPMT*1/*3A sample failed to amplify, and no whole blood was available for repeat DNA isolation. Conclusions: This method for rapid-cycle real-time, allele-specific amplification PCR-assisted long-range haplotyping has general application for the haplotyping of distant SNPs. The procedure is simpler and more rapid than previous methods. With respect to TPMT, haplotyping has the potential to discriminate the genotypes TPMT*1/*3A (intermediate metabolizer) and TPMT*3B/*3C (poor metabolizer).

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Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

Journal of Biochemical and Biophysical Methods ◽

10.1016/s0165-022x(02)00114-8 ◽

2003 ◽

Vol 55 (1) ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Michiyo Nagano ◽

Takahiro Nakamura ◽

Shogo Ozawa ◽

Keiko Maekawa ◽

Yoshiro Saito ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Long Range ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Sequencing Method ◽

Allele Specific ◽

Long Range Pcr

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Genomic Rearrangements at the BRCA1 Locus in Spanish Families with Breast/Ovarian Cancer

Clinical Chemistry ◽

10.1373/clinchem.2006.070110 ◽

2006 ◽

Vol 52 (8) ◽

pp. 1480-1485 ◽

Cited By ~ 49

Author(s):

Miguel de la Hoya ◽

Sara Gutiérrez-Enríquez ◽

Eladio Velasco ◽

Ana Osorio ◽

Ana Sanchez de Abajo ◽

...

Keyword(s):

Ovarian Cancer ◽

Real Time ◽

Long Range ◽

Real Time Pcr ◽

Genomic Rearrangements ◽

Brca1 And Brca2 ◽

Long Range Pcr ◽

Exon 11 ◽

Exon 20 ◽

Alternative Set

Abstract Background: Large genomic rearrangements (LGRs) account for a substantial proportion of the BRCA1 disease-causing changes, or variations, identified in families with hereditary breast/ovarian cancer [HB(O)C]. Great differences in the spectrum and prevalence of BRCA1 LGR have been observed among populations. Here we report the first comprehensive analysis of BRCA1 LGRs conducted in Spain. Methods: We used multiplex ligation-dependent probe amplification (MLPA) to screen for BRCA1 LGRs in the index case individuals of 384 HB(O)C families who previously tested negative for BRCA1 and BRCA2 point variations, small insertions, and deletions. An alternative set of MLPA probes, long-range PCR, and real-time PCR were used to confirm positive results. Results: We have identified 8 different BRCA1 rearrangements (del exon 1–24, del exon 8–13, del exon 11–15, del exon 14, dup exon 19–20, dup exon 20, exon 21–22 amplification, and del exon 23–24). With the exception of del exon 8–13, they are novel alterations. Overall, BRCA1 LGRs explain 1.4% of the Spanish HB(O)C families, and they account for 8.2% of all BRCA1 pathogenic variations identified in our study population. BRCA1 genetic variants affecting hybridization of commercially available MLPA probes are very rare in our population. Conclusions: Screening for BRCA1 LGRs should be mandatory in Spanish HB(O)C families. A high proportion of country-specific rearrangements are scattered along the gene. MLPA is a robust method to screen for LGRs in our population. MLPA analysis of positive samples with an alternative set of probes, together with long-range PCR and real-time PCR, is a feasible approach to confirm results in cases in which LGR breakpoints have not been characterized.

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Association of rs266729 and rs16861194 polymorphisms of the ADIPOQ gene with the risk of obesity in residents of the Moscow region

Almanac of Clinical Medicine ◽

10.18786/2072-0505-2021-49-038 ◽

2020 ◽

Vol 49 ◽

Author(s):

A. V. Pogozheva ◽

E. Yu. Sorokina

Keyword(s):

Real Time ◽

European Population ◽

Overweight And Obesity ◽

Minor Allele ◽

Moscow Region ◽

Real Time System ◽

Adipoq Gene ◽

Glucose Levels ◽

Allele Specific ◽

Specific Amplification

Rationale: The contribution of the adiponectin gene polymorphisms (ADIPOQ, located at 3q27) in the residents of Nigeria and China to the risk of overweight and its association with the risk of arterial hypertension in the European population have been demonstrated.Aim: To identify associations between rs266729 and rs16861194 polymorphisms of the ADIPOQ gene with overweight and obesity in the Moscow region residents.Materials and methods: Identification of rs266729 and rs16861194 polymorphisms of the ADIPOQ gene was carried out in 222 people (140 women and 82 men, aged 25 to 65 years) living in the Moscow region. Genotyping was performed using allele-specific amplification with real-time detection of the results on the CFX96 Real-Time System amplifier (Bio-Rad, USA) and using TaqMan probes complementary to polymorphic DNA regions. To assess an association between these genetic polymorphisms and overweight/obesity, we performed a case control study, with the cases being subjects with body mass index (BMI) of ≥ 30 kg/m2, and the controls those with BMI of < 30 kg/m2.Results: The mean frequency of the minor allele G rs266729 polymorphism of the ADIPOQ gene in the subjects from the Moscow region was 26.8% and similar in men and women. Comparison of the CC and GG genotypes carriers of the rs266729 polymorphism of the ADIPOQ gene in men showed a statistically significant association of the GG genotype to the BMI value (p = 0.04). There were no statistically significant differences between anthropometric indicators (BMI, body fat mass) in the carriers of different rs16861194 polymorphism genotypes of the ADIPOQ. No association between the studied polymorphisms and blood glucose levels and lipid spectrum could be found.Conclusion: The frequency of the minor allele G of the rs266729 polymorphism of the ADIPOQ gene in the subjects from the Moscow region was similar to their rates in the Russian Federation and European countries. In Moscow residents, the rs266729 polymorphism of the ADIPOQ gene (G allele) contributes to the risk of obesity in homozygous carriers (genotype GG). No association of the rs16861194 polymorphism of the ADIPOQ gene with the BMI was found; therefore, this polymorphism cannot be considered as a genetic marker of the obesity risk.

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Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

PLoS ONE ◽

10.1371/journal.pone.0153576 ◽

2016 ◽

Vol 11 (4) ◽

pp. e0153576 ◽

Cited By ~ 26

Author(s):

Alexandre Harlé ◽

Julia Salleron ◽

Claire Franczak ◽

Cindy Dubois ◽

Pierre Filhine-Tressarieu ◽

...

Keyword(s):

Next Generation Sequencing ◽

High Resolution ◽

Metastatic Melanoma ◽

Real Time ◽

High Resolution Melting ◽

Braf Mutations ◽

Allele Specific ◽

Specific Amplification ◽

Automated Platform ◽

Generation Sequencing

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Single Nucleotide Polymorphisms in the Chicken Mx gene at Position 2032 by Real-time Allele-specific PCR Melting-curve Analysis

The Journal of Poultry Science ◽

10.2141/jpsa.009070 ◽

2010 ◽

Vol 47 (2) ◽

pp. 133-138 ◽

Cited By ~ 1

Author(s):

Xiangqun Ye ◽

Zongcheng Tan ◽

Yueling Zhang ◽

Kangsheng Li

Keyword(s):

Single Nucleotide Polymorphisms ◽

Real Time ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

Disease Markers ◽

10.1155/2010/432462 ◽

2010 ◽

Vol 29 (5) ◽

pp. 243-249 ◽

Cited By ~ 1

Author(s):

Line Wee ◽

Hege Vefring ◽

Grete Jonsson ◽

Astanand Jugessur ◽

Rolv Terje Lie

Keyword(s):

Melting Curve ◽

Renin Angiotensin System ◽

Western World ◽

Melting Curve Analysis ◽

Nucleotide Polymorphisms ◽

Nucleotide Substitutions ◽

Hybridization Probe ◽

Hybridization Probes ◽

Specific Hybridization ◽

Allele Specific

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) inREN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

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Optimized Strategy for Rapid Cytochrome P450 2D6 Genotyping by Real-Time Long PCR

Clinical Chemistry ◽

10.1373/49.10.1624 ◽

2003 ◽

Vol 49 (10) ◽

pp. 1624-1631 ◽

Cited By ~ 31

Author(s):

Burkhardt Müller ◽

Konstanze Zöpf ◽

Julia Bachofer ◽

Werner Steimer

Keyword(s):

Cytochrome P450 ◽

Real Time ◽

Gel Electrophoresis ◽

Epidemiologic Studies ◽

Cytochrome P450 2D6 ◽

Long Pcr ◽

Hybridization Probes ◽

Pcr Product ◽

Working Day ◽

P450 2D6

Abstract Background: Because of genetic polymorphisms, cytochrome P450 2D6 (Cyp2D6) activity in humans varies widely and alters the metabolism of commonly used drugs such as antidepressants, neuroleptics, and cardioactive agents. Severe adverse effects or resistance to therapy may result. Methods: We performed long PCR on the LightCycler™ and used the product as a template for a previously validated multiplex PCR that examines the *3, *4, *6, *7, and *8 alleles of Cyp2D6. We used real-time PCR to identify the *5 null allele and duplication of Cyp2D6 with detection by either hybridization probes or SYBR Green®. The *2 −1584 C/G polymorphism and the *35 allele were identified by PCR with detection by hybridization probes. Products of all PCRs were visualized with gel electrophoresis using a 0.7–1.5% agarose gel and ethidium bromide. Samples containing the *35 allele were analyzed in parallel by digestion with NlaIII, MslI, and BstXI and SmaI. We analyzed samples from volunteers and patients (105 samples for deletion and duplication and 116 samples for preamplification). Of those samples, 59 were from depressive inpatients taking part in a trial not yet published. Results: Identical genotyping results for both real-time and conventional PCR were obtained and verified by gel electrophoresis. Use of long-PCR methods on the LightCycler enabled comprehensive analysis of all relevant polymorphisms of the Cyp2D6 gene in 1 working day with a hands-on time of ∼3–4 h. Conclusions: This is the first description of a successful long-PCR application on the LightCycler and the fastest technique for amplification and specific detection of a PCR product of comparable length. The method appears suitable for large clinical and epidemiologic studies.

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