Validating a Rapid Method for Detecting Common Polymorphisms in the APOA5 Gene by Melting Curve Analysis Using LightTyper

Francès, Francesc; Corella, Dolores; Sorlí, José V.; Guillén, Marisa; González, José I.; Portolés, Olga

doi:10.1373/clinchem.2005.049676

Cited by 16 publications

(10 citation statements)

References 14 publications

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“…which two or more peaks are observed at dissociation curve are non-specific amplifications, primer dimers, real-time multiplex PCR (Guion and Ochoa, 2008) (Frances et al, 2005). In this study, melting curve analysis showed two separate peaks at real-time PCR dissociation curve that was completely reproducible.…”

Section: Discussionsupporting

confidence: 49%

Causes of bimodal melting curve:Asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

Abtahi¹,

Sadeghi²,

Shabani³

et al. 2011

Afr. J. Biotechnol.

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The aim of this study was to present a new situation in which a relatively single short PCR-product might show two separate peaks with sequence specific shapes at the dissociation curve. SYBR-Green I real-time RT-PCR was performed on Lhcgr-gene transcripts in rats. Different programs were used for melting curve simulation and estimating Tm. Statistical tests were performed to determine whether two peaks at the dissociation curve were belonging to a single template. A bimodal melting curve was observed in real-time RT-PCR on a short segment (169 bp) of Lhcgr gene with a single band in gel electrophoresis. Sequencing of the Cloned PCR-product was compatible with template sequence. Realtime PCR using the vector conveying interested sequence, showed again two peaks at dissociation curve. The GC-content of first 100 bases (75%) and last 69 bases (42%) were significantly different. DNA melting simulation programs also confirmed the bimodal pattern, although, their height and wideness were different to actual peaks. Due to the asymmetric GC distribution effect on dissociation curve in short sequences, it is highly recommended to use DNA melting simulation programs to predict the number of peaks in the melting curve when designating primers; however, predicted peak shapes are not always accurate.

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Section: Discussionsupporting

confidence: 49%

Causes of bimodal melting curve:Asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

Abtahi¹,

Sadeghi²,

Shabani³

et al. 2011

Afr. J. Biotechnol.

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“…All PCR were performed in a Biometra thermal cycler, using Taq Polymerase (Bioline). A fragment of 157 bp including the 56C > G polymorphism was amplified using two oligonucleotides, forward: 5′- GGC TCT TCT TTC AGG TGG GTCBTCCG -3′reverse: 5′- GCC TTT CCG TGC CTG GGT GGT -3′ [9]. The PCR conditions were an initial denaturing at 96°C for 5 min, followed by 30 cycles of 96°C for 30 s, 64°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Association of APOA5 rs662799 and rs3135506 polymorphisms with arterial hypertension in Moroccan patients

et al. 2014

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BackgroundThe goal of the study is to investigate the association between the APOA5 polymorphisms and haplotypes with Arterial Hypertension (AHT) in Moroccan patients.MethodsThe study was performed in 283 subjects, 149 patients with AHT and 134 controls. All subjects were genotyped for the APOA5 -1131 T > C (rs662799), 56C > G (rs3135506) and c.553G > T (rs2075291) polymorphisms.ResultsThere was a strong association between -1131 T > C and 56C > G polymorphisms with AHT. The -1131 T > C and 56C > G polymorphisms were significantly associated with increased systolic blood pressure (SBP) and triglycerides (TG) levels. There were 4 haplotypes with a frequency higher than 5%, constructed from APOA5 polymorphisms, with the following order: -1131 T > C, 56C > G and c.553G > T. Haplotype H1 (TCG) was associated with decreased risk of AHT, whereas the haplotypes H2 (CCG) and H4 (CGG) were significantly associated with an increased risk of AHT. Carriers of H1 haplotype had a lower SBP and DBP and TG. In contrast, significant elevated SBP, DBP and TG were found in H4 haplotypes carriers.ConclusionsOur data demonstrate for the first time that several common SNPs in the APOA5 gene and their haplotypes are closely associated with modifications of blood pressure and serum lipid parameters in the AHT patient.

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“…Several single nucleotide polymorphisms (SNPs) were recently genotyped using FRET alone [2,8]) or in association with RFLP method and in these cases 100% concordance was found [3,6]. On the contrary, in our study due to the presence of samples S15 and S24, we had discrepant results that lowered the concordance rate between the FRET or sequencing and RFLP to 99.75%.…”

Section: Resultsmentioning

confidence: 52%

APOE genotyping: comparison of three methods

Rihn

Berrahmoune²,

Jouma

et al. 2008

Clin Exp Med

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Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, epsilon(2,) epsilon(3) and epsilon(4), and to identify in a single reaction any of the six following genotypes: epsilon(2)/epsilon(2), epsilon(3)/epsilon(3), epsilon(4)/epsilon(4), epsilon(3)/epsilon(4), epsilon(4)/epsilon(2), epsilon(3)/epsilon(2). The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for epsilon(2)/epsilon(2) and epsilon(4)/epsilon(4) homozygotes, (2) 4.0, 6.6 and 14.7% for epsilon(2)/epsilon(4), epsilon(2)/epsilon(3) and epsilon(3)/epsilon(4) heterozygotes, respectively, and (3) 72.0% for epsilon(3)/epsilon(3) homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.

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Validating a Rapid Method for Detecting Common Polymorphisms in the APOA5 Gene by Melting Curve Analysis Using LightTyper

Cited by 16 publications

References 14 publications

Causes of bimodal melting curve:Asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

Causes of bimodal melting curve:Asymmetric guanine-cytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

Association of APOA5 rs662799 and rs3135506 polymorphisms with arterial hypertension in Moroccan patients

APOE genotyping: comparison of three methods

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