{"title":"基因分型 SNP 和 Indels:提高基于高分辨率熔体 (HRM) 分析应用范围和灵敏度的方法。","authors":"","doi":"10.1016/j.cca.2024.119897","DOIUrl":null,"url":null,"abstract":"<div><p>High-resolution melt (HRM) analysis is a closed-tube technique for detecting single nucleotide polymorphisms (SNPs). However, it has limited use in high-resolution melting devices, even those with high thermal accuracy (HTA). In addition to the cost of switching to these specialized devices, the presence of nearest neighbour neutral changes (class III, IV SNPs and small indels) made HRM-based assays a challenging task due to reduced sensitivity. This study aimed to design a common modified competitive amplification of differently melting amplicons (CADMA)-based assay to address these challenges by generating allele-specific qPCR products that are detectable on most qPCR platforms.</p><p>For this study, SNPs were selected from all four classes of SNPs (class I: C/T or G/A mutation; class II: C/A or G/T mutation; class III: G/C mutation; class IV: A/T mutation). A single base pair and 19 bp indels were also chosen to simulate how CADMA primers could be designed for indels of varying lengths. The melting temperatures (Tm) were determined using IDT oligoAnalyzer. qPCR and melt data acquisition were performed on the CFX96 qPCR platform, and the melt curve data were analyzed using Precision Melt software (Bio-Rad, USA). The clusters for different genotypes were successfully identified with the aid of the control samples, and Tm predictions were carried out using the uMelt batch and Tm online tools for comparison.</p><p>Using HRM-qPCR assays based on the modified CADMA method, genotyping of various SNPs was successfully carried out. For some SNPs, similarly shaped melt curves were observed for homozygotes and heterozygotes, making shape-based genotype prediction difficult. The Tm values calculated via the Blake and Delcourts (1998) method were the closest to the experimental Tm values after adjusting for the salt concentration.</p><p>Since HRM assays usually depend on the ΔTm caused by mutations, they are prone to a high error rate due to nearest neighbour neutral changes. The technique developed in this study significantly reduces the failure rates in HRM-based genotyping and could be applied to any SNP or indel in any platform. It is crucial to have a deep understanding of the melt instrument, its accuracy and the nature of the target (SNP class or indel length and GC content of the flanking region). Furthermore, the availability of controls is essential for a high success rate.</p></div>","PeriodicalId":10205,"journal":{"name":"Clinica Chimica Acta","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Genotyping SNPs and Indels: A method to improve the scope and sensitivity of High-Resolution melt (HRM) analysis based applications\",\"authors\":\"\",\"doi\":\"10.1016/j.cca.2024.119897\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>High-resolution melt (HRM) analysis is a closed-tube technique for detecting single nucleotide polymorphisms (SNPs). However, it has limited use in high-resolution melting devices, even those with high thermal accuracy (HTA). In addition to the cost of switching to these specialized devices, the presence of nearest neighbour neutral changes (class III, IV SNPs and small indels) made HRM-based assays a challenging task due to reduced sensitivity. This study aimed to design a common modified competitive amplification of differently melting amplicons (CADMA)-based assay to address these challenges by generating allele-specific qPCR products that are detectable on most qPCR platforms.</p><p>For this study, SNPs were selected from all four classes of SNPs (class I: C/T or G/A mutation; class II: C/A or G/T mutation; class III: G/C mutation; class IV: A/T mutation). A single base pair and 19 bp indels were also chosen to simulate how CADMA primers could be designed for indels of varying lengths. The melting temperatures (Tm) were determined using IDT oligoAnalyzer. qPCR and melt data acquisition were performed on the CFX96 qPCR platform, and the melt curve data were analyzed using Precision Melt software (Bio-Rad, USA). The clusters for different genotypes were successfully identified with the aid of the control samples, and Tm predictions were carried out using the uMelt batch and Tm online tools for comparison.</p><p>Using HRM-qPCR assays based on the modified CADMA method, genotyping of various SNPs was successfully carried out. For some SNPs, similarly shaped melt curves were observed for homozygotes and heterozygotes, making shape-based genotype prediction difficult. The Tm values calculated via the Blake and Delcourts (1998) method were the closest to the experimental Tm values after adjusting for the salt concentration.</p><p>Since HRM assays usually depend on the ΔTm caused by mutations, they are prone to a high error rate due to nearest neighbour neutral changes. The technique developed in this study significantly reduces the failure rates in HRM-based genotyping and could be applied to any SNP or indel in any platform. It is crucial to have a deep understanding of the melt instrument, its accuracy and the nature of the target (SNP class or indel length and GC content of the flanking region). Furthermore, the availability of controls is essential for a high success rate.</p></div>\",\"PeriodicalId\":10205,\"journal\":{\"name\":\"Clinica Chimica Acta\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.2000,\"publicationDate\":\"2024-08-02\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Clinica Chimica Acta\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0009898124021508\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICAL LABORATORY TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinica Chimica Acta","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0009898124021508","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICAL LABORATORY TECHNOLOGY","Score":null,"Total":0}
Genotyping SNPs and Indels: A method to improve the scope and sensitivity of High-Resolution melt (HRM) analysis based applications
High-resolution melt (HRM) analysis is a closed-tube technique for detecting single nucleotide polymorphisms (SNPs). However, it has limited use in high-resolution melting devices, even those with high thermal accuracy (HTA). In addition to the cost of switching to these specialized devices, the presence of nearest neighbour neutral changes (class III, IV SNPs and small indels) made HRM-based assays a challenging task due to reduced sensitivity. This study aimed to design a common modified competitive amplification of differently melting amplicons (CADMA)-based assay to address these challenges by generating allele-specific qPCR products that are detectable on most qPCR platforms.
For this study, SNPs were selected from all four classes of SNPs (class I: C/T or G/A mutation; class II: C/A or G/T mutation; class III: G/C mutation; class IV: A/T mutation). A single base pair and 19 bp indels were also chosen to simulate how CADMA primers could be designed for indels of varying lengths. The melting temperatures (Tm) were determined using IDT oligoAnalyzer. qPCR and melt data acquisition were performed on the CFX96 qPCR platform, and the melt curve data were analyzed using Precision Melt software (Bio-Rad, USA). The clusters for different genotypes were successfully identified with the aid of the control samples, and Tm predictions were carried out using the uMelt batch and Tm online tools for comparison.
Using HRM-qPCR assays based on the modified CADMA method, genotyping of various SNPs was successfully carried out. For some SNPs, similarly shaped melt curves were observed for homozygotes and heterozygotes, making shape-based genotype prediction difficult. The Tm values calculated via the Blake and Delcourts (1998) method were the closest to the experimental Tm values after adjusting for the salt concentration.
Since HRM assays usually depend on the ΔTm caused by mutations, they are prone to a high error rate due to nearest neighbour neutral changes. The technique developed in this study significantly reduces the failure rates in HRM-based genotyping and could be applied to any SNP or indel in any platform. It is crucial to have a deep understanding of the melt instrument, its accuracy and the nature of the target (SNP class or indel length and GC content of the flanking region). Furthermore, the availability of controls is essential for a high success rate.
期刊介绍:
The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells.
The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.