TILLING,即Targeting Induced Local Lesions IN Genomes(定向诱导基因组局部突变技术),是由美国Fred Hutchinson癌症研究中心Steven Henikoff领导的研究小组发展起来的,是一种全新的反向遗传学研究方法。TILLING技术借助高通量的检测手...TILLING,即Targeting Induced Local Lesions IN Genomes(定向诱导基因组局部突变技术),是由美国Fred Hutchinson癌症研究中心Steven Henikoff领导的研究小组发展起来的,是一种全新的反向遗传学研究方法。TILLING技术借助高通量的检测手段,快速有效地从由化学诱变剂(EMS)诱变过的突变群体中鉴定出点突变。目前,TILLING已被应用于多种生物的研究中。本文系统介绍了TILLING技术的基本原理、技术路线及其技术优势,同时列举了TILLING的应用实例,并展望了TILLING技术的应用前景。展开更多
BACKGROUND The aim of this study was to investigate the complex heterozygous mutations of ANK1 and SPTA1 in the same individual and improve our understanding of hereditary spherocytosis(HS)in children.We also hope to ...BACKGROUND The aim of this study was to investigate the complex heterozygous mutations of ANK1 and SPTA1 in the same individual and improve our understanding of hereditary spherocytosis(HS)in children.We also hope to promote the application of gene detection technology in children with HS,with the goals of identifying more related gene mutations,supporting the acquisition of improved molecular genetic information to further reveal the pathogenesis of HS in children,and providing important guidance for the diagnosis,treatment,and prevention of HS in children.CASE SUMMARY A 1-year and 5-month-old patient presented jaundice during the neonatal period,mild anemia 8 months later,splenic enlargement at 1 year and 5 months,and brittle red blood cell permeability.Genetic testing was performed on the patient,their parents,and sister.Swiss Model software was used to predict the protein structure of complex heterozygous mutations in ANK1 and SPTA1.Genetic testing revealed that the patient harbored a new mutation in the ANK1 gene from the father and a mutation in the SPTA1 gene from the mother.Combined with the clinical symptoms of the children,it is suggested that the newly discovered complex heterozygous mutations of ANK1 and SPTA1 may be the cause,providing important guidance for revealing the pathogenesis,diagnosis,treatment,and promotion of gene detection technology in children with HS.CONCLUSION This case involves an unreported complex heterozygous mutation of ANK1 and SPTA1,which provides a reference for exploring HS.展开更多
Background:Leber’s Hereditary Optic Neuropathy(LHON;MIM 535000)is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset betwe...Background:Leber’s Hereditary Optic Neuropathy(LHON;MIM 535000)is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15–30.The worldwide incidence of LHON is approximately 1 in 31,000.95%of LHON patients will have one of 3 primary mitochondrial mutations,G3460A(A52T of ND1),G11778A(R340H of ND4)and T14484C(M64V of ND6).There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50%of male carriers and 10%of female carriers developing optic neuropathy.Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation.The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism(RFLP)or individual targeted bi-directional Sanger sequencing reactions.The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay.Methods:PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site.A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay.Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls.DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay.The RFLP products were detected by agarose gel electrophoresis.Results:The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure.The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations.Conclusion:In this paper,we describe a nove展开更多
文摘TILLING,即Targeting Induced Local Lesions IN Genomes(定向诱导基因组局部突变技术),是由美国Fred Hutchinson癌症研究中心Steven Henikoff领导的研究小组发展起来的,是一种全新的反向遗传学研究方法。TILLING技术借助高通量的检测手段,快速有效地从由化学诱变剂(EMS)诱变过的突变群体中鉴定出点突变。目前,TILLING已被应用于多种生物的研究中。本文系统介绍了TILLING技术的基本原理、技术路线及其技术优势,同时列举了TILLING的应用实例,并展望了TILLING技术的应用前景。
基金Supported by The Science and Technology Department of Sichuan Province,No.2021JDKP0015.
文摘BACKGROUND The aim of this study was to investigate the complex heterozygous mutations of ANK1 and SPTA1 in the same individual and improve our understanding of hereditary spherocytosis(HS)in children.We also hope to promote the application of gene detection technology in children with HS,with the goals of identifying more related gene mutations,supporting the acquisition of improved molecular genetic information to further reveal the pathogenesis of HS in children,and providing important guidance for the diagnosis,treatment,and prevention of HS in children.CASE SUMMARY A 1-year and 5-month-old patient presented jaundice during the neonatal period,mild anemia 8 months later,splenic enlargement at 1 year and 5 months,and brittle red blood cell permeability.Genetic testing was performed on the patient,their parents,and sister.Swiss Model software was used to predict the protein structure of complex heterozygous mutations in ANK1 and SPTA1.Genetic testing revealed that the patient harbored a new mutation in the ANK1 gene from the father and a mutation in the SPTA1 gene from the mother.Combined with the clinical symptoms of the children,it is suggested that the newly discovered complex heterozygous mutations of ANK1 and SPTA1 may be the cause,providing important guidance for revealing the pathogenesis,diagnosis,treatment,and promotion of gene detection technology in children with HS.CONCLUSION This case involves an unreported complex heterozygous mutation of ANK1 and SPTA1,which provides a reference for exploring HS.
文摘Background:Leber’s Hereditary Optic Neuropathy(LHON;MIM 535000)is one of the most commonly inherited optic neuropathies and it results in significant visual morbidity among young adults with a peak age of onset between the ages of 15–30.The worldwide incidence of LHON is approximately 1 in 31,000.95%of LHON patients will have one of 3 primary mitochondrial mutations,G3460A(A52T of ND1),G11778A(R340H of ND4)and T14484C(M64V of ND6).There is incomplete penetrance and a marked gender bias in the development of visual morbidity with approximately 50%of male carriers and 10%of female carriers developing optic neuropathy.Visual recovery can occur but is dependent on the mutation present with the highest level of visual recovery seen in patients who have the T14484C mutation.The 3 primary mutations are typically identified by individual end-point PCR-restriction fragment length polymorphism(RFLP)or individual targeted bi-directional Sanger sequencing reactions.The purpose of this study was to design a simple multiplex PCR-RFLP that could detect these 3 primary LHON mutations in one assay.Methods:PCR primers were designed to incorporate a MaeIII restriction site in the presence of 3460A and 14484C mutations with the 11778A mutation naturally incorporating a MaeIII site.A multiplex PCR-RFLP assay was developed to detect the 3 common mutations in a single assay.Synthetic LHON controls based on the mitochondrial genome harbouring the 3 common mutations were synthesized and cloned into plasmids to act as reliable assay controls.DNA from previously tested patients and the synthetic LHON controls were subjected to the multiplex PCR-RFLP assay.The RFLP products were detected by agarose gel electrophoresis.Results:The novel PCR-RFLP assay accurately detects the 3 primary mutations both in patient DNA and in synthesized DNA control samples with a simple visual mutation detection procedure.The synthesized DNA was demonstrated to be a robust control for the detection of LHON Mutations.Conclusion:In this paper,we describe a nove
