Marino M A, Devaney J M, Davis P A, Smith J K, Girard J E
Operational Genetics Laboratory, Armed Forces Institute of Pathology, Washington, D.C. 20306, USA.
Anal Chem. 1998 Nov 1;70(21):4514-9. doi: 10.1021/ac980526q.
Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.
短串联重复序列（STR）等位基因因其高度多态性而被广泛用作法医标记。通常通过凝胶电泳将它们分离，并用嵌入染料进行可视化。本研究的目的是确定DNA-嵌入染料复合物的吸光度和荧光随碱基对（bp）与染料比例的变化。DNA样本由来自THO1、F13A01和vWFA31位点的STR等位基因组成。这些等位基因通过PCR扩增并经HPLC纯化，以确保每个样本中仅存在所需的DNA片段。等位基因大小范围从vWFA位点的151 bp（等位基因17）到F13A01位点的199 bp（等位基因8）。腺嘌呤和胸腺嘧啶（AT）含量从THO1位点的48%到F13A01和vWFA31位点的69%不等。每个位点的纯合等位基因分别与双嵌入剂TOTO-1和YOYO-1及其相应的单体染料TOPRO-1和YOPRO-1混合。随着每种嵌入染料的加入，DNA-染料复合物在260 nm处的吸光度增加。减去染料吸光度后，DNA在260 nm处的吸光度保持恒定。当单体和二聚体染料嵌入DNA螺旋时，荧光发射显著增加。对于所有三个位点，在双嵌入剂TOTO-1的碱基对与染料比例为10/1和YOYO-1的碱基对与染料比例为5/1时，观察到荧光强度平台。使用AT浓度最高的F13A01位点的等位基因8与嵌入剂YOYO-1获得了最大的荧光强度响应。
