A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

Abstract

Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

“…In the conventional CE system, separation of the signals is enabled using probes of different lengths . However, when probe lengths are varied, false‐negative results often arise because of nonspecific reactions due to higher melting temperatures or G/C contents of some target probes or due to the possible formation of secondary structures . Therefore, CE‐SSCP, a conformation‐sensitive CE system, was introduced in our method to avoid the problematic use of different‐length probes that would have been necessary using the conventional method.…”

Multiplex ligase‐based genotyping methods combined with CE

“…The sieving medium, an amphiphilic triblock copolymer (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)), is capable of forming a stable network of uniformly sized micelles that are packed into a cubic structure [71]. By applying this novel polymer matrix to ligated products, similarsized products could achieve baseline separation [72][73][74][75][76][77][78]. In the cited studies, to create unique electrophoretic migrations after conventional MLPA procedures, the stufferfree probes could be analyzed simultaneously without further postprocessing.…”

“…Because neither stuffers nor mobility modifiers are involved in this separation process, the variation was termed as stuffer-free MLPA. Stuffer-free MLPA provides a more precise quantita-tive analysis compared to conventional MLPA, because the method is free from the problems that originate from the size variation between the probes [74,77]. Despite the strong potential, stuffer-free MLPA has limitations because of a deficiency in the fundamental understanding of the separation principle.…”

Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation

“…CE-SSCP was used to detect the amplified probes; this method is suitable for analyzing multiple probes using a single fluorescent tag. Unlike conventional CE, which requires the probes to have different lengths 21 , 22 , CE-SSCP can separate DNA molecules based on conformational differences arising from sequence diversity. In particular, using Pluronic polymer as the separation matrix will dramatically increase the resolution, so that up to 20 targets can be simultaneously analyzed in a single assay 21 – 25 .…”