DNA中点突变检测方案(大分子作用仪)

HORIBAJOBIN YVONSPRi & Life Sciences SPRi & Life SciencesApplication Note SPRi-0707.3-v1-Page 2 Point Mutations of DNA Sequences Michael CANVA-Laboratoire Charles Fabry， Institut dOptique， CNRS， UMR 8501， Bat 503， Centre scientifique， 91403 Orsay， France Genes can be mutated in several different ways. The simplest type of mutation involves a change in a single basealong the sequence of a particular gene (much like a typographical error in a word that has been misspelled). Inother instances， one or more bases may be added or deleted. Sometimes too， segments of a DNA molecule areaccidentally repeated， deleted or moved. DNA mutations generate genetic disorders that are responsible for hereditary diseases and have a predominantrole in many diseases such as cancers. Using GenOptics SPRi sensitive technology (Surface Plasmon Resonance imaging)a method was developed todetect DNA point mutations. The gold sensor surface (the glass prism) was covered witha mixed self-assembled multilayer based on electrostaticinteractions made of 3 layers of MUA： 11 Mercapto-undecanoic acid， PEl： poly(ethyleimine) and ExtrAvidin.Matrices composed of 100 or 196 spots (10 x 10 or 14 x 14matrices) were prepared and data from allIIpparallelhybridisation kinetics was collected. As this technique is highly sensitive and very precise itenables accurate identification of mutations even at a singlebase mismatch in an oligonucleotide sequence. lmage a： Image of the gold surface bearing 100 spots distributed on anarea of 16 mm?， having reacted with an oligonucleotide mixturereproducing the conditions of a patient genotype. In addition， the recognition specificity of functionalized spotsis such that each spot reacts only with the relevantCcomplementary sequence within a mixture of differentsequences.FForr example， b/y reconstituting patientsgenotypes with appropriate oligonucleotide mixtures (homoor heterozygote)， it is possible to determine their genotypewith no ambiguity. The results of the experiments are published in Sensorsaand Actuators， 2003，B94， 313-323. Fig. 1： From the image we observe kinetics associated with spots reactingrespectively to a complementary sequence (4R2)， presenting a mutation(4R1) or non relevant (AR=0). The system makes it possible to monitor simultaneouslyseveral hundred measurements， and additionally， directlycompare the effects induced by the variation of one orseveral parameters..An example is given in Figure 2，showing the variations of an hybridisation signal betweenprobe and target of various oligonucleotide lengths. Only one gene microtiter plate was prepared with differentgenotypic sequences grouped in pairs of various length presenting pairwise mutations (either by mutationl ordeletion). A mixture of target oligonucleotides including oneof the two (wild or mutated) species， corresponding to eachof the pairs was injected in the detection cell. Using this technique， it is possible to quantify in a single runall kinetics corresponding to an entire column of the sensormatrix， and this for each sequence studied. Fig. 2： Hybridisation kinetics recorded during 20 minutes periods. Rows show targets of various lengths France： +33(0)16454 13 00USA： +1 7324948660 Japan： +81 (0)3 3861 8231Germany： +49 (0)89 46 23 17-0UK：+44(0)20 8204 8142 Italy： +39 0 2 5760 3050China： +86(0)108567 9966Other Countries： +33 (0)1 64 54 13 00 HORIBAExplore the future Find us at www.jobinyvon.com/SPRi or telephone：HORIBAExplore the future Using GenOptics SPRi sensitive technology (Surface Plasmon Resonance imaging) a method was developed to detect DNA point mutations.