Development of a Standard Control for qMSP to Analyze the Methylation Status of LINE-1

Abstract

Cancer screening is an important aspect of comprehensive health care, making a significant contribution to reducing the risk of death and the cost of treating patients. Finding new tumor markers with high sensitivity and specificity is a trend in the research activities in Vietnam as well as all over the world. Long interspersed element-1 (LINE-1), a large proportion of repeating DNA, is transposable to different positions in the genome. LINE-1 activity is controlled through DNA methylation (the CpG is attached with CH₃), whereby LINE-1 is highly methylated in normal cells. However, in some types of cancer such as lung, breast, stomach, liver, rectum... changes in the methylation status LINE-1 have been noticed. To study the methylation status of the LINE-1 sequence, we used a quantitative methyl-specific PCR technique. This method requires a standard calibrator to quantify the rate of methylation. From the commercial methylated DNA (CpG Methylated Human Genomic DNA, Promega), we cloned two regions of the LINE-1 promoter corresponding to a reference sequence and an investigated one (target sequence) which are bisulfite transformed. As a result, we have created two recombinant plasmids, pRef-LINE1 and pMe-LINE1. The plasmids were mixed at 10% of methylation and could be used as a standard control for analyzing the DNA methylation of specimens from patients.

Keywords: DNA methylation, DNA repeating sequence, LINE-1, quantitative methyl-specific-PCR (qMSP), tumor markers.

References

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