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Maria J. Sanchez James M. Bradeen 《Molecular breeding : new strategies in plant improvement》2006,17(2):137-148
Resistance (R) genes and the proteins they encode are key components of the defense system of plants. The exploration of R
gene diversity enables the study of R gene evolution and may facilitate the isolation of new and functional alleles. Most
cloned R genes occur in clusters of related sequences. Thus, the development of a tool for reliable recovery of orthologous
R gene sequences to the exclusion of paralogous sequences will facilitate R gene diversity analysis. The late blight resistance
gene RB is a single functional locus embedded within a cluster of related sequences. Previously, the functional RB allele was cloned from wild potato using a Long Range-PCR (LR-PCR) technique, suggesting this method may be a promising tool
for recovery of R gene orthologs in other genotypes. Using the RB gene as a model, we explored the limitations and improved three technical aspects of LR-PCR for multi-genotype applications.
We present improved primers for the recovery of the RB locus and have identified efficient DNA extraction procedures and reliable amplification systems. We document that consensus
sequences built from three independently generated LR-PCR clones can be up to 100% accurate. Our results show encouraging
advances toward successful application of LR-PCR for isolating alleles from orthologous R gene loci. 相似文献
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Xiao-Ping Qi Zhen-Fang Du Ju-Ming Ma Xiao-Ling Chen Qing Zhang Jun Fei Xiao-Ming Wei Dong Chen Hai-Ping Ke Xuan-Zhu Liu Feng Li Zhen-Guang Chen Zheng Su Hang-Yang Jin Wen-Ting Liu Yan Zhao Hu-Ling Jiang Zhang-Zhang Lan Peng-Fei Li Ming-Yan Fang Wei Dong Xian-Ning Zhang 《Gene》2013
Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by genetic and allelic heterogeneity, large multi-exon genes, and duplication sequences of PKD1. Recently, targeted resequencing by pooling long-range polymerase chain reaction (LR-PCR) amplicons has been used in the identification of mutations in ADPKD. Despite its high sensitivity, specificity and accuracy, LR-PCR is still complicated. We performed whole-exome sequencing on two unrelated typical Chinese ADPKD probands and evaluated the effectiveness of this approach compared with Sanger sequencing. Meanwhile, we performed targeted gene and next-generation sequencing (targeted DNA-HiSeq) on 8 individuals (1 patient from one family, 5 patients and 2 normal individuals from another family). Both whole-exome sequencing and targeted DNA-HiSeq confirmed c.11364delC (p.H3788QfsX37) within the unduplicated region of PKD1 in one proband; in the other family, targeted DNA-HiSeq identified a small insertion, c.401_402insG (p.V134VfsX79), in PKD2. These methods do not overcome the screening complexity of homology. However, the true positives of variants confirmed by targeted gene and next-generation sequencing were 69.4%, 50% and 100% without a false positive in the whole coding region and the duplicated and unduplicated regions, which indicated that the screening accuracy of PKD1 and PKD2 can be largely improved by using a greater sequencing depth and elaborate design of the capture probe. 相似文献
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Purpose
Matrix Gla protein (MGP) is a molecular determinant regulating the extracellular matrix calcification. To further confirm whether the MGP genetic polymorphism was universally associated with the risk of kidney stone, we investigated the association of genetic polymorphisms of MGP with kidney stone in the Chinese Han population.Materials and methods
728 subjects were recruited for the study. We firstly re-sequenced the human genomic MGP gene including the 1500 bp promoter, 5′-UTR, 4 exons and 3′-untranslated regions, identified single nucleotide polymorphisms (SNPs) in MGP, and performed an association analysis with kidney stones in 54 subjects of the Chinese Han population. A candidate tag SNP was genotyped in total subjects using an allele specific PCR, and further analyzed the association with kidney stone.Results
We identified 18 polymorphisms including four tag SNPs. A tag SNPrs4236 was associated with kidney stones. The G allele carrier had a 1.373-fold reduced kidney stone risk compared with A allele carriers in SNPrs4236 (odds ratios (OR) = 1.373; 95%CI, 1.051–1.793; p = 0.019). However, we did not find an association between the polymorphism and clinical characteristics of kidney stones.Conclusions
Our findings showed that SNPrs4236 of the MGP gene is associated with kidney stones in the Chinese Han population, and influences the genetic susceptibility to kidney stones. In the future, functional assays of the polymorphism should permit a better understanding of the role of MGP genetic variants and kidney stones. 相似文献5.
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