核酸分析设计及分析.pptVIP

常规PCR引物及杂交探针设计 Primer 3 软件介绍 /primer3/input.htm Primer-BLAST A tool for finding specific primers 定量RT-PCR SYBR Green I 是一种结合于小沟中的双链 DNA 结合染料。与双链 DNA 结合后，其荧光大大增强。 Taqman技术原理是探针分子分别标记荧光和荧光淬灭集团，利用Taq酶的5′外切酶活性，裂解双链，将探针5′端连接的荧光分子从探针上切割下来，从而发出荧光。 molecular beacon分子信标是分别在5′和3′端的荧光标记物质和荧光淬灭剂。5′和3′构成探针的茎部。形成茎 环发夹结构，使荧光剂和淬灭剂紧密接触，导致荧光淬灭。杂交使探针5′和3′端分离，淬灭剂对荧光剂的淬灭作用消失，产生荧光。 Lightcycler技术。将荧光分子和淬灭分子分别标记在两个不同的探针上，生成荧光发生探针和淬灭探针，杂交时两探针的荧光分子和淬灭分子紧密相邻，从而发生FRET使荧光淬灭。 根据mRNA序列设计RT-PCR 引物 设计需要考虑的问题： 1、引物和探针的特异性 2、引物扩增的效果 3、扩增产物在mRNA的位置 4、能否鉴别基因组DNA的污染 DNA甲基化特异性PCR引物设计 亚硫酸氢钠修饰DNA的目的是将DNA序列中未甲基化的胞嘧啶完全转化为尿嘧啶，而甲基化的5-甲基胞嘧啶保持不变。 MSP需要特殊要求： 1。甲基化引物、非甲基化引物.Tm差异不能太大， 2。甲基化引物和非甲基化引物带有至少一个CpG序列，越多甲基化位点越好。至少有一个CpG序列在引物3‘端。最好设计一对野生型引物来验证摸板。3。M和U同时出现条带，说明这个甲基化位点是半甲基化的，或者你的模板没修饰完全。4。MSP的可靠性，最终还是要用测序来知道 RNAi targete selection rule 1.Targeted regions on the cDNA sequence of a targeted gene should be located 50-100 nt downstream of the start codon (ATG). 2.Search for sequence motif AA(N19)TT or NA(N21), or NAR(N17)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U). 3.Avoid targeting introns, since RNAi only works in the cytoplasm and not within the nucleus. 4.Avoid sequences with 50% G+C content. 5.Avoid stretches of 4 or more nucleotide repeats. 6.Avoid 5URT and 3UTR, although siRNAs targeting UTRs have successfully induced gene inhibition. 7.Avoid sequences that share a certain degree of homology with other related or unrelated genes. siRNA Database Searchable database of Silencer ? Validated and Pre-designed siRNAs to 34,000 human, mouse, and rat targets. All siRNAs in the database have been designed for maximum potency and specificity using a well-tested, proprietary algorithm . And silencing results are guaranteed. miRNA 数据库检索及实验设计 Micro (mi)RNAs are small, highly conserved noncoding RNAs that control gene expression post-transcriptionally either via the degradation of target mRNAs or the inhibition of protein translation. Each miRNA is believed to reg