Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter

Abstract

Methylation-specific polymerase chain reaction (PCR) (MSP) is frequently used to study gene silencing by promoter hypermethylation. However, non-specific primer design can lead to false-positive detection of methylation. We present a novel, web-based algorithm for the design of primers for bisulfite-PCRs (MSP, sequencing, COBRA and multiplex-MSP), allowing the determination of a specificity score, which is based on the thermodynamic characteristics of the primer 3 0 -end. PCR amplification with primers not reaching a high specificity score can result in false-positive findings. We used MSPprimer to design MSP primers for analysis of the ATM promoter. In 37 non-small cell lung cancer (NSCLC) samples and 43 breast cancer samples no promoter methylation was detected. Conversely, published MSP primers not reaching the required specificity score led to non-specific amplification of DNA not converted by bisulfite. The result was a false-positive incidence of ATM promoter methylation of 24% in NSCLC and 48% in breast cancers, similar to published studies. This highlights the critical need for specific primer design for MSP. MSPprimer is a convenient tool to achieve this goal, which is available free of charge to the scientific community.

Methylation-specific PCR: four steps in primer design

“…Initially, default settings of the programs described above were used. These results showed that: i) some primers designed by Methprimer have low ability to differentiate between methylated and unmethylated alleles ( Figure 5A), which is in line with the study done by Brandes et al [39], ii) BiSearch designed primers should be used only in the case of p14 and MGMT, and iii) primers provided by Methylprimer express have short sequences (18-22 nt). Therefore, it is not suprising that primer pairs specific for unmethylated alleles have low Tm.…”

“…Brandes and coworkers [39] showed that the role of ATM methylation is probably overestimated in breast and NSCLC samples. They found that results from previous publications were based on primers lacking specificity.…”

“…However, Methprimer is not without drawbacks. For example, Tm overestimation of AT rich primers has been reported [39]. There is also the principal objection that the algorithm behind Methprimer is only a modification of the Primer 3 software [40].…”

Biomarkers for detection and prognosis of breast cancer identified by a functional hypermethylome screen

“…For MSP analysis, DNA was extracted following standard phenol-chloroform extraction and bisulfite modified using the EZ DNA methylation Kit TM (Zymo Research). Primer sequences were designed using MSPPrimer 31 and Methylation-specific PCR was …”

Expression profiling identifies new function of collapsin response mediator protein 4 as a metastasis-suppressor in prostate cancer

“…According to the bisulfate-sequencing PCR results, the MSP primer pairs for CRMP4 were designed complying with the standard rules (Li and Dahiya, 2002;Brandes et al, 2007) to detect the methylation status of CpG island 1. The first step and second step PCR were amplified, as the same as bisulfate-sequencing PCR.…”