Tối ưu hóa quy trình kĩ thuật PCR phân tích một số exon trên gen CPT2 và SLC25A20=Optimization of the PCR technical process for analyzing of some exons on the CPT2 and SLC25A20 genes

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Tác giả: Thị Loan Nguyễn, Thị Phương Thúy Nguyễn, Văn Thạo Tạ, Đức Minh Trần, Thị Thúy Lành Trần

Ngôn ngữ: vie

Ký hiệu phân loại:

Thông tin xuất bản: Tạp chí Y học Việt Nam (Tổng hội Y học Việt Nam), 2024

Mô tả vật lý: tr.41-47

Bộ sưu tập: Metadata

ID: 243822

Thêm vào giỏ

Bệnh thiếu hụt Carnitine palmitoyltransferase 1 (CPT2) và bệnh thiếu hụt Carnitine acylcarnitine translocase (CACT) là những bệnh lý di truyền lặn trên nhiễm sắc thể thường. Nguyên nhân chính thường do đột biến các gen mã hóa cho các enzyme tham gia trình β oxi hóa acid béo, dẫn tới thiếu hụt những enzyme này. Kỹ thuật sinh học phân tử giải trình tự gen Sanger (Sanger sequencing) hiện đã được ứng dụng để chẩn đoán xác định đột biến gen CPT2/SLC25A20 do tính chính xác và đặc hiệu cao hơn các phương pháp xét nghiệm hóa sinh truyền thống. Trong giải trình tự gen Sanger, giai đoạn khuếch đại đoạn gen đặc hiệu bằng PCR có yêu cầu khắt khe về nhiệt độ gắn mồi, nồng độ mồi hay số chu kỳ nhiệt để đảm bảo chất lượng các đoạn gen thu được sau phản ứng. Nghiên cứu này tập trung vào tối ưu hóa bộ mồi đặc hiệu trong xác định đột biết gen CPT2 và SLC25A20. Kết quả cho thấy nhiệt độ gắn mồi từ 53°C – 57°C và nồng độ mồi trong khoảng 0,5 μmol/L – 1,0 μmol/L là tối ưu cho đa số các exon trong phân tích hai gen trên. Số chu kỳ nhiệt tối ưu là 40 chu kỳ với DNA khuôn được tách từ máu toàn phần có nồng độ 78,3 ng/μL.Carnitine palmitoyltransferase 2 (CPT2) deficiency and Carnitine acylcarnitine translocase (CACT) deficiency are autosomal recessive genetic disorders. The primary cause is typically mutations in genes encoding enzymes involved in the β-oxidation pathway of fatty acids, leading to deficiencies in these enzymes. Molecular biology techniques such as PCR or sequencing have been employed for accurate diagnosis of mutations in the CPT2/SLC25A20 genes, offering higher specificity compared to traditional biochemical tests. In Sanger sequencing, the stage of amplifying specific gene segments by PCR has strict requirements on primer annealing temperature, primer concentration, or number of thermal cycles to ensure the quality of gene segments obtained after the reaction. This study focuses on optimizing specific primer sets for the detection of mutations in the CPT2 and SLC25A20 genes. The results indicated that a primer annealing temperature ranging from 53°C to 57°C and a primer concentration between 0,5 μmol/L to 1,0 μmol/L are optimal for analyzing most exons of the two genes. The optimal thermal cycling conditions consisted of 40 cycles using genomic DNA extracted from whole blood with a concentration of 78,3 ng/μL.

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