一个引物找周边序列1.进 NCBI BLAST 模式2.粘贴相关序列 blast 后（例如水稻内源基因引物 GGAAAGCCGATGGCATCAG）3.出现相关序列4.找到下方alignment,也就是Download ” GEnBfink: <3「日ohicsOryza sativa Japonica Group 0s06g0604400 (0s06g0604400) mRNA; complete cdsSeq Lie nee ID::他胴陌_001064552.11 Length: 3144 Numbe『of Matches: 1Range 1: 1750 to 1758 GenBmnk Graphics T Next Match A Previous MatchScoreExpectIdentitiesGapsStrand38.2 bits(19}0.6619/19(100%)0/19(0%)Plus/PlusQuery 1GGAAAGCCGAT GGCATCAG 19Sbjct 1750GGAAAGCCGlkT GGCATCAG 1763HDownload ” Ge门亦nk OaphicsOryza sativa Japonica Group genomic DNA, chromosome 6, PAC clone:P0481H08Sequenee ID:: dbjj|AP0Q36293： Length: 146841 Number of IMatclies: 1Range 1: 27905 to 27923 WnBmrlc Graphics T Next Match A Previous MatchScore Expect Identities Gaps Strand38.2 bits(19) 0・66 19/19(100%) 0/1 今(0%) Phs/PlusQuery 1 GG?kAAGCCGATGGCATCAG 19I I I I I I I I I M I H I H I ISbj ct 27905 GGAAAGCCGATGGCATCAG 279235.选择合适的物种，点击graphics，得到下面图形最下方的序列为我们输进去的序列，上面两条黑色的链条是与其匹配的DNA双链，点击并 按住鼠标可左右移动该双链，点击右键可以下载（FASTA格式为单纯序列格式），下载的序 列片段长度为网页所显示的长度，如果想扩大范围，可以点击移动双链序列上方的（-+）按1 - +|27,920严,920 、一对引物找扩增序列（电子 PCR）以水稻内源基因引物为例:上游外引物 F3-2: GGAAAGCCGATGGCATCAG下游外引物 B3-2: TCTCCATTGTCCTCCTCTGC（反向互补 GCAGAGGAGGACAATGGAGA）进 NCBI BLAST 页面，找到下面的特殊 blast 中的 primer-blastBasic BLASTChoose a BLAST program to run.nucleotide blastSearch a nucleotide database using a nucleotide queryAlgorithms: blastn, megablast disc on tiguous megablastprotein blastSearch protein database using a protein query Algorithms: blastp, psi-blast, phi-blast, delta-blastblastxSearch protein database using a translated nucleotide querytblastnSearch translated nucleotide database using a protein querytblastxSearch translated nucleotide database using a translated nucleotide qiSpecialized BLASTChoose a type of specialized search (or database name in parentheses.)口 Get faster protein results with a graphical view using SmartBLAST 口 Make specific primers with P「ime「~BLAST点开 primer-blast 页面A.输入自己的引物B. 选择数据库 database （如 Mrna,或 genomes）C. 输入物种（范围可大可小，如 plant 植物）Primer Pair Specificity Checking ParametersSpecificity check翻 Enable search for primer pairs specific to ttiie intended PCR template 妙Search modeAutomaticDatabaseRefseq mRNAOrganismvascular plants (taxi±58023)FntAr ah nrnanism nams fnr nmanism nrniin namft such ae RntRmhactRriACfiasD.点^卩讪，就可以出结果。
Sequence (5'->3')LengthTmGC%Self complen*Forward primerGGAAAGCCGATGGCATCAG195&9757.897.00Reverse primerTCTCCATTGTCCTCCTCTGC2058.8055.003.00Products on target templates'NM—OOI 064552.1 Oryza sativa Japonica Group 0s06g0604400 (0s06g0604400) mRNA, complete cdsproduct length = 198Forward primer 1 GGAAAGCCGATGGCATCAG 19Template 1750 1768Reverse primer 1 TCTCCATTGTCCTCCTCTGC 20Template 1947 1928>XM_006656157.1 PREDICTED: Oryza brachyantha phospholipase D alpha 2>like (LOC102714193), mRNAproduct Forward Templatelength primer二 19811720GGAAAGCCGATGGCATCAG191738ReverseTemplateprimer11917TCTCCATTGTCCTCCTCTGC C 201898得全基因图；了 Orvza sativa Japonica Group 0s06a0604400 E.点击合适物种链接如 NCBI Reference Sequence: NM C010&4552 1F.也可以继续点击graphics^壬 ，得下图（点击-+按键调节显示序列，点击并按住可以拖动双链至合适位置（D上显示1750-1928为目的 序列）：12001400180080012闫纠gI罗四I胡goI苓goEi IGenes,001064552.1: 1.7K..2.0K(255bp)Oryza sativa Japonica Group 0s06g0604400 (0s06g0604400) mRNA, complete cdsNCBI Reference Sequence: NM_001064552.1 GenBank FASTALink To This Pane | FeedbackG.在双链上点击右键，选save，选fasta,然后保存到电脑，此fa文件可以用IE打开，得到 下列目的序列>gi| 1 1 5 468 835 |ref|NM_0 0 1 0645 5 2 . 1 |: 1 7 4 1 - 1 966 Oryza sativa Japonica Group Os06g0604400 (Os06g0604400) mRNA, complete cdsCATTTGCATGGAAAGCCGATGGCATCAGACCAGAAGACATTGAGGCGTTGCATCTGATTCCCAGAGAGATTTCTCTGAAGATTGTGAACAAGATTGAAGCTGGTGAGCGTTTTGCAGTCTATGTTGTGCTGCCAATGT GGCCTGAAGGACCTCCTGCTAGTGGATCAGTGCAGGCAATACTGGATTGGCAGAGGAGGACAATGGAGA TGATGTACTATGATATTGC3.水稻内源基因LAMP扩增片段及引物 水稻内源基因引物2:上游外引物 F3-2: GGAAAGCCGATGGCATCAG下游外引物 B3-2: TCTCCATTGTCCTCCTCTGC上 游 内 引 物 FIP-2 ： GCAAAACGCTCACCAGCTTCAA ( 反 向 互 补TTGAAGCTGGTGAGCGTTTTGC) -GACATTGAGGCGTTGCATCT下游内引物 BIP-2： ATGTTGTGCTGCCAATGTGGC-CCAGTATTGCCTGCACTGAT （反向互补 ATCAGTGCAGGCAATACTGG）内源基因内源基因扩增序列如下：CATTTGCATGGAAAGCCGATGGCATCAGACCAGAAGACATTGAGGCGTTGCATCTGATTCCCAGAGAGA TTTCTCTGAAGATTGTGAACAAGATTGAAGCTGGTGAGCGTTTTGCAGTCTATGTTGTGCTGCCAATGT GGCCTGAAGGACCTCCTGCTAGTGGATCAGTGCAGGCAATACTGGATTGGCAGAGGAGGACAATGGAGA TGATGTACTATGATATTGC。