Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers
Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers
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DOI：
10.2144/05382st03
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发表时间：
2005-02-01
期刊：
影响因子：
2.7
通讯作者：
da Silva, ACR
中科院分区：
文献类型：
--
作者：
Levano-Garcia, J;Verjovski-Almeida, S;da Silva, ACR
建立了一种基于降落PCR的转座子插入位点定位新方法，该方法利用一种混合共有简并引物和一种与转座子退火的特异性引物相结合的方法来确定转座子在基因组DNA中的插入位点。该方法使用柑橘黄单胞菌转座子突变体进行了测试。PCR产物含有两个X.柑橘基因组DNA和转座子。仅使用特异性引物从PCR直接测序产物。不同的PCR条件进行了测试，并优化的反应参数，提高产品的产量和特异性进行了描述。用HIBI 7杂合引物获得最佳结果，HIBI 7杂合引物是在3'末端的最后12个碱基内的6个不同位置具有简并碱基的25-mer寡核苷酸。X利用EZ：：TN转座子随机转座构建了柑橘突变体文库，并鉴定了90个突变体基因组内的插入位点。在这些X染色体和质粒DNA中都发现了插入。柑橘变种限制性酶切图谱和Southern印迹分析证实了8个随机选择的突变体的插入位点。该方法为功能基因组学研究中突变体的大规模表征提供了一个非常有用的工具。
A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomona.s citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIBI7 hybrid primer which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.