Optimizing methodologies for PCR-based DNA methylation analysis

Hernández, Hernán Guillermo; Tse, M. Yat; Pang, Stephen C.; Arboleda, Humberto; Forero, Diego A.

doi:10.2144/000114087

Cited by 153 publications

(123 citation statements)

References 76 publications

(134 reference statements)

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“…Unmethylated sequences are usually amplified more than methylated sequences, particularly at lower temperatures; smaller amplicons and sequences with fewer CpG sites have higher amplification efficiencies than longer amplicons containing more CpG sites (Wojdacz and Hansen, 2006;Hernández et al, 2013). Primer affinity to its binding site may be manipulated by design and annealing temperature adjustments (Wojdacz and Hansen, 2006;Wojdacz et al, 2008Wojdacz et al, , 2009).…”

Section: Discussionmentioning

confidence: 99%

“…Among the methodologies for methylation pattern access, of note are methylationspecific polymerase chain reaction (PCR), bisulfite sequencing, and methylation-sensitive melting curves analysis (MS-MCA) (Hernández et al, 2013). MS-MCA is an attractive approach to quantification analyses because it is inexpensive and allows several samples to be analyzed simultaneously (Worm et al, 2001;Wojdacz and Hansen, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…This induces DNA sequence differences that depend on the initial methylation status. Therefore, methylated sequences have greater numbers of C and G than unmethylated sequences, producing a difference in the melting curve profiles (Worm et al, 2001;Tost, 2009;Hernández et al, 2013). Owing to the evolution of intercalating dye and real-time PCR platforms, melting curve analyses have been termed high resolution melting (HRM) (Tost, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes

Carobin

et al. 2015

Genet. Mol. Res.

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ABSTRACT. High-resolution melting (HRM) is considered an inexpensive, rapid, and attractive methodology for methylation analysis. In the application of the polymerase chain reaction (PCR) to methylation analysis, amplification efficiencies are biased towards unmethylated, rather than methylated, templates: a phenomenon known as PCR bias. To overcome PCR bias, primers that include CpG site(s) and are fully complementary to the methylated sequence have been proposed. However, genes mapped within imprinted regions usually present higher methylation levels, and an unusual PCR bias towards the methylated template can therefore arise. The manipulation of primer affinity attempts to overcome this problem. We attempted to show that mismatches at the primer's methylated binding sites increase the area between the 50 and 100% methylation plots on the melting curves, and may increase HRM accuracy for samples that have high methylation levels. Sets of primers for imprinted genes that included CpG sites at their binding sequences were designed, and were complementary to methylated or unmethylated templates. Primers fully complementary 7865 Manipulation of primer affinity and imprinted genes ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 7864-7872 (2015) to methylated templates produced a very small area between the 50 and 100% methylation plots. When using primers that were fully complementary to the unmethylated sequence, we were able to increase the area between the 50 and 100% methylation plots. Therefore, when samples are highly methylated, such as targets in genes mapped in imprinted regions, we propose that primers should favor amplification of the rarest, unmethylated sequence. Primers may be designed to include one CpG at its binding site and be fully complementary to the unmethylated template.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes

Carobin

et al. 2015

Genet. Mol. Res.

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“…These factors determine "restrictive points", i.e., sequences where primers should not be located. For example, it is recommended to avoid sequences containing common SNPs [30]. Also, promoter regions having a high level of homology with other sequences in the genome should also be avoided.…”

Section: Where Should Primers Be Located?mentioning

confidence: 99%

“…The optimal choice would eliminate the main drawback of the method: the possibility that obtained results are false positives [30]. False positive results are the consequence of incomplete bisulfite modification, or low ability of the methylated primer pair to differentiate between methylated and unmethylated alleles.…”

Section: Rules and Software For Primer Designmentioning

confidence: 99%

Methylation-specific PCR: four steps in primer design

et al. 2014

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Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.

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High-Resolution Melting Analysis for Identifying Sequence Variations in Nuclear Genes for Assembly Factors and Structural Subunits of Cytochrome C Oxidase

Vondráčková

Veselá

Zeman

et al. 2015

Methods in Molecular Biology

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High-resolution melting (HRM) analysis is a simple, sensitive, and cost-effective screening method. HRM enables the detection of homozygous or heterozygous point sequence variants and small deletions within specific PCR products by observing temperature and shape changes in melting curve profiles using fluorescent dyes. Herein, an updated protocol for routine variant screening of nuclear genes encoding assembly factors and structural subunits of cytochrome c oxidase (COX) is described. Nonetheless, the general recommendations given for HRM analysis can be applicable for examining any genetic region of interest.

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Optimizing methodologies for PCR-based DNA methylation analysis

Cited by 153 publications

References 76 publications

Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes

Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes

Methylation-specific PCR: four steps in primer design

High-Resolution Melting Analysis for Identifying Sequence Variations in Nuclear Genes for Assembly Factors and Structural Subunits of Cytochrome C Oxidase

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