Kashkin K N, Strizhkov B N, Griadunov D A, Surzhikov S A, Grechishnikova I V, Kreĭndlin E Ia, Chupeeva V V, Evseev K B, Turygin A Iu, Mirzabekov A D
Mol Biol (Mosk). 2005 Jan-Feb;39(1):30-9.
We combined three modern technologies of single base polymorphism detection in human genome: ligase detection reaction, rolling circle amplification and IMAGE hydro-gel microarrays. Polymorphism in target DNA was tested by selective ligation on microarray. Product of the ligase reaction was determined in microarray gel pads by rolling circle amplification. Two different methods were compared. In first, selective ligation of short oligonucleotides immobilized on microarray was used with subsequent amplification on preformed circle probe ("common circle"). The circle probe was designed especially for human genome research. In second variant, allele-specific padlock probes that may be circularized by selective ligation were immobilized on microarray. Polymorphism of codon 72 in human p53 gene was used as a biological model. It was shown that LDR/RCA on microarray is a quantitative reaction and gives high discrimination of alleles. Principles and perspectives of selective ligation and rolling circle amplification are being discussed.
连接酶检测反应、滚环扩增和IMAGE水凝胶微阵列。通过在微阵列上进行选择性连接来检测目标DNA中的多态性。连接酶反应的产物通过滚环扩增在微阵列凝胶垫中进行测定。比较了两种不同的方法。第一种方法是使用固定在微阵列上的短寡核苷酸进行选择性连接，随后在预先形成的环状探针（“通用环”）上进行扩增。该环状探针是专门为人类基因组研究设计的。在第二种变体中，可通过选择性连接环化的等位基因特异性锁式探针固定在微阵列上。人类p53基因第72密码子的多态性被用作生物学模型。结果表明，微阵列上的连接酶检测反应/滚环扩增是一种定量反应，能够对等位基因进行高度区分。文中还讨论了选择性连接和滚环扩增的原理及前景。
