Maulik G, Botchway S, Chakrabarti S, Tetradis S, Price B, Makrigiorgos G M
Dana Farber Cancer Institute, Joint Center for Radiation Therapy, Department of Radiation Oncology,Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA.
Nucleic Acids Res. 1999 Mar 1;27(5):1316-22. doi: 10.1093/nar/27.5.1316.
A highly sensitive method to detect traces of aldehyde-containing apurinic/apyrimidinic (AP) sites in nucleic acids has been developed. Based on this method, a novel approach to detect DNA base mismatches recognized by the mismatch repair glycosylase MutY is demonstrated. Open chain aldehydes generated in nucleic acids due to spontaneous depurination, DNA damage or base excision of mismatched adenine by MutY are covalently trapped by a new linker molecule [fluorescent aldehyde-reactive probe (FARP), a fluorescein-conjugated hydroxylamine derivative]. DNA containing AP sites is FARP-trapped, biotinylated and immobilized onto neutravidin-coated microplates. The number of FARP-trapped aldehydes is then determined via chemiluminescence using a cooled ICCD camera. AP sites induced in plasmid or genomic calf thymus DNA via mild depurination or by simple incubation at physiological conditions (pH 7, 37 degreesC) presented a linear increase in chemiluminescence signal with time. The procedure developed, from a starting DNA material of approximately 100 ng, allows detection of attomole level (10(-18) mol) AP sites, or 1 AP site/2 x 10(7) bases, and extends by 1-2 orders of magnitude the current limit in AP site detection. In order to detect MutY-recognized mismatches, nucleic acids are first treated with 5 mM hydroxylamine to remove traces of spontaneous aldehydes. Following MutY treatment and FARP-labeling, oligonucleotides engineered to have a centrally located A/G mismatch demonstrate a strong chemiluminescence signal. Similarly, single-stranded M13 DNA that forms mismatches via self-complementation (average of 3 mismatches over 7429 bases) and treated with MutY yields a signal approximately 100-fold above background. No signal was detected when DNA without mismatches was used. The current development allows sensitive, non-isotopic, high throughput screening of diverse nucleic acids for AP sites and mismatches in a microplate-based format.
已开发出一种高灵敏度方法来检测核酸中痕量含醛的脱嘌呤/脱嘧啶（AP）位点。基于此方法，展示了一种检测由错配修复糖基化酶MutY识别的DNA碱基错配的新方法。由于自发脱嘌呤、DNA损伤或MutY对错配腺嘌呤的碱基切除而在核酸中产生的开链醛，被一种新的连接分子[荧光醛反应探针（FARP），一种荧光素共轭羟胺衍生物]共价捕获。含有AP位点的DNA被FARP捕获、生物素化并固定在中性抗生物素蛋白包被的微孔板上。然后使用冷却的ICCD相机通过化学发光测定FARP捕获的醛的数量。通过温和脱嘌呤或在生理条件（pH 7，37℃）下简单孵育在质粒或基因组小牛胸腺DNA中诱导的AP位点，其化学发光信号随时间呈线性增加。从大约100 ng的起始DNA材料开始开发的该程序，能够检测阿托摩尔水平（10⁻¹⁸ mol）的AP位点，即1个AP位点/2×10⁷个碱基，并将当前AP位点检测的极限扩展了1 - 2个数量级。为了检测MutY识别的错配，首先用5 mM羟胺处理核酸以去除痕量的自发醛。在MutY处理和FARP标记之后，设计为在中心位置具有A/G错配的寡核苷酸显示出强烈的化学发光信号。同样，通过自我互补形成错配（在7429个碱基上平均有3个错配）并经MutY处理的单链M13 DNA产生的信号比背景高约100倍。当使用没有错配的DNA时未检测到信号。当前的进展允许以微孔板形式对各种核酸中的AP位点和错配进行灵敏、非同位素、高通量筛选。
