【求助】请高手看看我的引物设计
这个帖子发布于 19 年零 177 天前，其中的信息可能已发生改变或有所发展。
我要设计mouse LAG-3的引物，在pubmed 上查到的序列是，
ORIGIN
1 gggcagtggg gaggagaagc agaaggactg ggtctggagg agcagctcaa gttctagcta
61 gctgcagtgg gtttgcctgc actctgctct gggtcccagc ccgggcctct gatcattatc
121 catcctgctg tctccagtcc ccactcctgg ggcgtcctct tcaccctaca ttctttccct
181 ccgcctcacc tcctccttgt agaacttctc tctctctctc tctctctctc tctctctctc
241 tctctctctc tctctgtgtg tgtgtgtgtg tctgtctgtc tgtctctctc tctctcctcc
301 caggaccttt ttctaacctc ccttggaggg ctggggaggc ccgggccata gaggagatga
361 gggaggacct gctccttggc tttttgcttc tgggactgct ttgggaagct ccagttgtgt
421 cttcagggcc tgggaaagag ctccccgtgg tgtgggccca ggagggagct cccgtccatc
481 ttccctgcag cctcaaatcc cccaacctgg atcctaactt tctacgaaga ggaggggtta
541 tctggcaaca tcaaccagac agtggccaac ccactcccat cccggccctt gaccttcacc
601 aggggatgcc ctcgcctaga caacccgcac ccggtcgcta cacggtgctg agcgtggctc
661 caggaggcct gcgcagcggg aggcagcccc tgcatcccca cgtgcagctg gaggagcgcg
721 gcctccagcg cggggacttc tctctgtggt tgcgcccagc tctgcgcacc gatgcgggcg
781 agtaccacgc caccgtgcgc ctcccgaacc gcgccctctc ctgcagtctc cgcctgcgcg
841 tcggccaggc ctcgatgatt gctagtccct caggagtcct caagctgtct gattgggtcc
901 ttttgaactg ctccttcagc cgtcctgacc gcccagtctc tgtgcactgg ttccagggcc
961 agaaccgagt gcctgtctac aactcaccgc gtcatttttt agctgaaact ttcctgttac
1021 tgccccaagt cagccccctg gactctggga cctggggctg tgtcctcacc tacagagatg
1081 gcttcaatgt ctccatcacg tacaacctca aggttctggg tctggagccc gtagcccctc
1141 tgacagtgta cgctgctgaa ggttctaggg tggagctgcc ctgtcatttg cccccaggag
1201 tggggacccc ttctttgctc attgccaagt ggactcctcc tggaggaggt cctgagctcc
1261 ccgtggctgg aaagagtggc aattttaccc ttcaccttga ggctgtgggt ctggcacagg
1321 ctgggaccta cacctgtagc atccatctgc agggacagca gctcaatgcc actgtcacgt
1381 tggcggtcat cacagtgact cccaaatcct tcgggttacc tggctcccgg gggaagctgt
1441 tgtgtgaggt aaccccggca tctggaaagg aaagatttgt gtggcgtccc ctgaacaatc
1501 tgtccaggag ttgcccgggc cctgtgctgg agattcagga ggccaggctc cttgctgagc
1561 gatggcagtg tcagctgtac gagggccaga ggcttcttgg agcgacagtg tacgccgcag
1621 agtctagctc aggcgcccac agtgctagga gaatctcagg tgaccttaaa ggaggccatc
1681 tcgttctcgt tctcatcctt ggtgccctct ccctgttcct tttggtggcc ggggcctttg
1741 gctttcactg gtggagaaaa cagttgctac tgagaagatt ttctgcctta gaacatggga
1801 ttcagccatt tccggctcag aggaagatag aggagctgga gcgagaactg gagacggaga
1861 tgggacagga gccggagccc gagccggagc cacagctgga gccagagccc aggcagctct
1921 gacctggagc cgaggcagcc agcaggtctc agcagctccg cccgcccgcc cgcccgcccg
1981 aataaactcc ctgtcagcag cat
用Primer５进行引物设计的结果如下，
1. 5' CCGACTTAGGGTACAAGATTC 3'
rating 94, Seq No:197, Length:21, Tm:53.6, GC%:47.6%, activity[ug/OD]31.6 ,Product size:414, Ta Opt 54.9, Degeneracy: 1.0.
3' CGAAGGATTTGGGAGTCACT
rating 100, Seq No:610, Length:20, Tm:54.4, GC%:50% , activity[ug/OD]31.7, Product size:474, Ta Opt 54.7, Degeneracy: 1.0.
sense 发现有Dimer 和cross Dimer。anti-sense没有。
根据引物设计的原理有Dimer 和cross Dimer都不好，但这是rating最高的一条引物，请高手看看是否能改进？如何改进？
ORIGIN
1 gggcagtggg gaggagaagc agaaggactg ggtctggagg agcagctcaa gttctagcta
61 gctgcagtgg gtttgcctgc actctgctct gggtcccagc ccgggcctct gatcattatc
121 catcctgctg tctccagtcc ccactcctgg ggcgtcctct tcaccctaca ttctttccct
181 ccgcctcacc tcctccttgt agaacttctc tctctctctc tctctctctc tctctctctc
241 tctctctctc tctctgtgtg tgtgtgtgtg tctgtctgtc tgtctctctc tctctcctcc
301 caggaccttt ttctaacctc ccttggaggg ctggggaggc ccgggccata gaggagatga
361 gggaggacct gctccttggc tttttgcttc tgggactgct ttgggaagct ccagttgtgt
421 cttcagggcc tgggaaagag ctccccgtgg tgtgggccca ggagggagct cccgtccatc
481 ttccctgcag cctcaaatcc cccaacctgg atcctaactt tctacgaaga ggaggggtta
541 tctggcaaca tcaaccagac agtggccaac ccactcccat cccggccctt gaccttcacc
601 aggggatgcc ctcgcctaga caacccgcac ccggtcgcta cacggtgctg agcgtggctc
661 caggaggcct gcgcagcggg aggcagcccc tgcatcccca cgtgcagctg gaggagcgcg
721 gcctccagcg cggggacttc tctctgtggt tgcgcccagc tctgcgcacc gatgcgggcg
781 agtaccacgc caccgtgcgc ctcccgaacc gcgccctctc ctgcagtctc cgcctgcgcg
841 tcggccaggc ctcgatgatt gctagtccct caggagtcct caagctgtct gattgggtcc
901 ttttgaactg ctccttcagc cgtcctgacc gcccagtctc tgtgcactgg ttccagggcc
961 agaaccgagt gcctgtctac aactcaccgc gtcatttttt agctgaaact ttcctgttac
1021 tgccccaagt cagccccctg gactctggga cctggggctg tgtcctcacc tacagagatg
1081 gcttcaatgt ctccatcacg tacaacctca aggttctggg tctggagccc gtagcccctc
1141 tgacagtgta cgctgctgaa ggttctaggg tggagctgcc ctgtcatttg cccccaggag
1201 tggggacccc ttctttgctc attgccaagt ggactcctcc tggaggaggt cctgagctcc
1261 ccgtggctgg aaagagtggc aattttaccc ttcaccttga ggctgtgggt ctggcacagg
1321 ctgggaccta cacctgtagc atccatctgc agggacagca gctcaatgcc actgtcacgt
1381 tggcggtcat cacagtgact cccaaatcct tcgggttacc tggctcccgg gggaagctgt
1441 tgtgtgaggt aaccccggca tctggaaagg aaagatttgt gtggcgtccc ctgaacaatc
1501 tgtccaggag ttgcccgggc cctgtgctgg agattcagga ggccaggctc cttgctgagc
1561 gatggcagtg tcagctgtac gagggccaga ggcttcttgg agcgacagtg tacgccgcag
1621 agtctagctc aggcgcccac agtgctagga gaatctcagg tgaccttaaa ggaggccatc
1681 tcgttctcgt tctcatcctt ggtgccctct ccctgttcct tttggtggcc ggggcctttg
1741 gctttcactg gtggagaaaa cagttgctac tgagaagatt ttctgcctta gaacatggga
1801 ttcagccatt tccggctcag aggaagatag aggagctgga gcgagaactg gagacggaga
1861 tgggacagga gccggagccc gagccggagc cacagctgga gccagagccc aggcagctct
1921 gacctggagc cgaggcagcc agcaggtctc agcagctccg cccgcccgcc cgcccgcccg
1981 aataaactcc ctgtcagcag cat
用Primer５进行引物设计的结果如下，
1. 5' CCGACTTAGGGTACAAGATTC 3'
rating 94, Seq No:197, Length:21, Tm:53.6, GC%:47.6%, activity[ug/OD]31.6 ,Product size:414, Ta Opt 54.9, Degeneracy: 1.0.
3' CGAAGGATTTGGGAGTCACT
rating 100, Seq No:610, Length:20, Tm:54.4, GC%:50% , activity[ug/OD]31.7, Product size:474, Ta Opt 54.7, Degeneracy: 1.0.
sense 发现有Dimer 和cross Dimer。anti-sense没有。
根据引物设计的原理有Dimer 和cross Dimer都不好，但这是rating最高的一条引物，请高手看看是否能改进？如何改进？
