tailieunhanh - Xây dựng đối chứng chuẩn cho kĩ thuật qMSP xác định tỉ lệ methyl hóa promoter LINE-1

Bài viết Xây dựng đối chứng chuẩn cho kĩ thuật qMSP xác định tỉ lệ methyl hóa promoter LINE-1 thiết kế được 2 cặp mồi khuếch đại trình tự tham chiếu cho LINE-1 và trình tự đích nhằm khảo sát tình trạng methyl hóa LINE-1. Bằng kĩ thuật tách dòng chúng tôi đã tạo ra được hai plasmid tái tổ hợp pRefLINE1 và pMe-LINE1 chứa đoạn DNA chuẩn đại diện cho hai trình tự trên của LINE-1 bị methyl hóa sau khi biến đổi bisulfite. | VNU Journal of Science Medical and Pharmaceutical Sciences Vol. 38 No. 1 2022 65-73 Original Article Development of a Standard Control for qMSP to Analyze the Methylation Status of LINE-1 Phạm Anh Thuy Duong Pham The Tung Tran Thi Quynh Trang Luu Thu Phuong Vo Thi Thuong Lan VNU University of Science 334 Nguyen Trai Thanh Xuan Hanoi Vietnam Received 03 May 2021 Revised 6 September 2021 Accepted 9 September 2021 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. _ Corresponding author. E-mail address vothithuonglan@ https 2588-1132 65