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Specificity of methylation assays in cancer research: a guideline for designing primers and probes.

Barekati Z, Radpour R, Kohler C, Zhong XY - Obstet Gynecol Int (2010)

Bottom Line: DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers.However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test.The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Gynecological Oncology, Women's Hospital and Department of Biomedicine, University of Basel, Basel 4031, Switzerland.

ABSTRACT
DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers. A large number of cancer studies have been focused on identification of methylation changes as biomarkers (i.e., breast cancer). However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test. This highlights the critical need for specific primer and probe design to avoid false-positive detection of methylation profiling. The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step.

No MeSH data available.


Related in: MedlinePlus

Primer design for DNA methylation profiling techniques based on bisulfite conversion. (a) First DNA is treated with sodium bisulfite to convert all unmethylated cytosines to uracil. To analyze DNA methylation status of the interest genes, converted DNA is amplified based on two different primer designing strategies: methylation-independent specific PCR (MIP) and methylation-specific PCR (MSP). (b) In MIP, DNA molecules are amplified using primer pairs containing cytosines (no-CpG) in their sequence. (c) In MSP, primer pairs are designed to specifically amplify either methylated (M) or unmethylated (U) DNA by containing CpG site in their sequence that makes possible to distinguish the methylated sequence from the unmethylated sequence.
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fig1: Primer design for DNA methylation profiling techniques based on bisulfite conversion. (a) First DNA is treated with sodium bisulfite to convert all unmethylated cytosines to uracil. To analyze DNA methylation status of the interest genes, converted DNA is amplified based on two different primer designing strategies: methylation-independent specific PCR (MIP) and methylation-specific PCR (MSP). (b) In MIP, DNA molecules are amplified using primer pairs containing cytosines (no-CpG) in their sequence. (c) In MSP, primer pairs are designed to specifically amplify either methylated (M) or unmethylated (U) DNA by containing CpG site in their sequence that makes possible to distinguish the methylated sequence from the unmethylated sequence.

Mentions: Primers should not contain any CpG sites within their sequence to avoid discrimination against methylated or unmethylated DNA (Figure 1).


Specificity of methylation assays in cancer research: a guideline for designing primers and probes.

Barekati Z, Radpour R, Kohler C, Zhong XY - Obstet Gynecol Int (2010)

Primer design for DNA methylation profiling techniques based on bisulfite conversion. (a) First DNA is treated with sodium bisulfite to convert all unmethylated cytosines to uracil. To analyze DNA methylation status of the interest genes, converted DNA is amplified based on two different primer designing strategies: methylation-independent specific PCR (MIP) and methylation-specific PCR (MSP). (b) In MIP, DNA molecules are amplified using primer pairs containing cytosines (no-CpG) in their sequence. (c) In MSP, primer pairs are designed to specifically amplify either methylated (M) or unmethylated (U) DNA by containing CpG site in their sequence that makes possible to distinguish the methylated sequence from the unmethylated sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2926695&req=5

fig1: Primer design for DNA methylation profiling techniques based on bisulfite conversion. (a) First DNA is treated with sodium bisulfite to convert all unmethylated cytosines to uracil. To analyze DNA methylation status of the interest genes, converted DNA is amplified based on two different primer designing strategies: methylation-independent specific PCR (MIP) and methylation-specific PCR (MSP). (b) In MIP, DNA molecules are amplified using primer pairs containing cytosines (no-CpG) in their sequence. (c) In MSP, primer pairs are designed to specifically amplify either methylated (M) or unmethylated (U) DNA by containing CpG site in their sequence that makes possible to distinguish the methylated sequence from the unmethylated sequence.
Mentions: Primers should not contain any CpG sites within their sequence to avoid discrimination against methylated or unmethylated DNA (Figure 1).

Bottom Line: DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers.However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test.The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Gynecological Oncology, Women's Hospital and Department of Biomedicine, University of Basel, Basel 4031, Switzerland.

ABSTRACT
DNA methylation is an epigenetic regulation mechanism of genomic function, and aberrant methylation pattern has been found to be a common event in many diseases and human cancers. A large number of cancer studies have been focused on identification of methylation changes as biomarkers (i.e., breast cancer). However, still clinical use of them is very limited because of lack of specificity and sensitivity for diagnostic test. This highlights the critical need for specific primer and probe design to avoid false-positive detection of methylation profiling. The guideline and online web tools that are introduced in this paper might help to perform a successful experiment and to develop specific diagnosis biomarkers by designing right primer pair and probe prior to experimental step.

No MeSH data available.


Related in: MedlinePlus