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gene121木虫 (著名写手)
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【资源】PCR引物银行的使用
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PCR引物银行是目前国际上最大的引物数据库，里面有超过18万种引物序列，它的引物序列能用于一般 的PCR，也能用于定量PCR，你只要输入基因针对的ID号（可以通过NCBI查询），这样就能得出引物序列。甚至你也可输入基因的名称，或蛋白质的名称，或者对应的Locallink都可以查到。目前引物主要针对人和小鼠的，今后将拓展到其它物种，如大鼠等。 它的引物成功率达到99%，我想大家应该放心使用了吧。 这个文章来源于一篇华人的文章 Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154; pp.1-8. 数据库地址： http://pga.mgh.harvard.edu/primerbank/ 或者：这是一个压缩文件，下载后解压缩即可使用，很方便！ 这个数据库的引物设计的规则如下： All the primers in PrimerBank were designed using a program called uPrimer. Great care has been given to avoid primer mispriming to other known genes in a genome. Here is a list of criteria for gene specific primer design: The primer length range: 19 - 23 nt, with the optimal length at 21 nt. The primer GC percentage range: 35% - 65%. The delta G value for the five 3’ end-bases is at least -9 kcal/mol. The primer Tm range: 60 - 63 °C, determined by the Nearest Neighbor Method. The PCR product length range is 100 - 250 bp. If this requirement cannot be satisfied, alternative ranges will be used. The default number of primer pairs designed for each sequence is 3. No primer is designed from low-complexity regions. A primer does not contain 6 or more contiguous same nucleotides. A primer does not contain any ambiguous nucleotide. No repetitive 15-mer from other gene sequences in the genome (for both strands) anywhere in a primer. No repetitive 13-mer from non-coding RNA sequences (for both strands) anywhere in a primer. The global BLAST score for any primer is less than 30 (equivalent to 15-mer perfect match). The maximum Tm for the 3’ end perfect match to other gene sequences does not exceed 46 °C; does not exceed 42 °C when compared to non-coding RNA sequences (Tm determined by the Nearest Neighbor Method). For primer secondary structure (the primer-primer self-annealing) No repetitive 5-mer is allowed anywhere when a primer sequence is compared to its complementary strand. The four 3’-end bases should be unique when compared to the primer’s complementary strand. The forward and reverse primers should not anneal to each other. The filter setting is the same as in the primer secondary structure filter. For sequence secondary structure (the primer-sequence self-annealing) No repetitive 9-mer is allowed when a primer sequence is compared to the complementary strand of its cognate sequence. The BLAST score is less than 18 when a primer sequence is compared to the complementary strand of its cognate sequence 这里是一个新的专门针对realtime PCR引物设计的数据库。 因为realtime PCR引物一般较短，因此设计方法与一般的PCR有些不同。这个数据库非常强大，包含的物种种类十分多。总共包括1071种基因的引物。 http://medgen.ugent.be/rtprimerdb/ 基因种类分部： Homo sapiens: 756 Mus musculus: 159 Rattus norvegicus: 141 Drosophila melanogaster: 5 Danio reri 2 Bos taurus: 8 Caenorhabditis elegans: 0 Human immunodeficiency virus 1: 0 Xenopus laevis: 0 Strongylocentrotus purpuratus: 0 Gallus gallus: 0 Sus scrofa: 0 Xenopus tropicalis: 0 Canis familiaris: 0 Total: 1071 |
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