Abstract
In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper, we report a modification of the realtime quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM™- and VIC®-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 210-219 |
| Number of pages | 10 |
| Journal | BioTechniques |
| Volume | 40 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2006 |
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ASJC Scopus subject areas
- Clinical Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
Cite this
Two-color quantitative multiplex methylation-specific PCR. / Swift-Scanlan, Theresa; Blackford, Amanda; Argani, Pedram; Sukumar, Saraswati; Fackler, Mary Jo.
In: BioTechniques, Vol. 40, No. 2, 02.2006, p. 210-219.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Two-color quantitative multiplex methylation-specific PCR
AU - Swift-Scanlan, Theresa
AU - Blackford, Amanda
AU - Argani, Pedram
AU - Sukumar, Saraswati
AU - Fackler, Mary Jo
PY - 2006/2
Y1 - 2006/2
N2 - In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper, we report a modification of the realtime quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM™- and VIC®-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.
AB - In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper, we report a modification of the realtime quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM™- and VIC®-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.
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UR - http://www.scopus.com/inward/citedby.url?scp=33645528784&partnerID=8YFLogxK
U2 - 10.2144/000112097
DO - 10.2144/000112097
M3 - Article
C2 - 16526411
AN - SCOPUS:33645528784
VL - 40
SP - 210
EP - 219
JO - BioTechniques
JF - BioTechniques
SN - 0736-6205
IS - 2
ER -