High resolution melting analysis of almond SNPs derived from ESTs

High resolution melting curve (HRM) is a recent advance for the detection of SNPs. The technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between samples. It has been applied to the analysis and scan of mutations in the genes causing human diseases. In plant species, the use of this approach is limited. We applied HRM analysis to almond SNP discovery and genotyping based on the predicted SNP information derived from the almond and peach EST database. Putative SNPs were screened from almond and peach EST contigs by HRM analysis against 25 almond cultivars. All 4 classes of SNPs, INDELs and microsatellites were discriminated, and the HRM profiles of 17 amplicons were established. The PCR amplicons containing single, double and multiple SNPs produced distinctive HRM profiles. Additionally, different genotypes of INDEL and microsatellite variations were also characterised by HRM analysis. By sequencing the PCR products, 100 SNPs were validated/revealed in the HRM amplicons and their flanking regions. The results showed that the average frequency of SNPs was 1:114 bp in the genic regions, and transition to transversion ratio was 1.16:1. Rare allele frequencies of the SNPs varied from 0.02 to 0.5, and the polymorphic information contents of the SNPs were from 0.04 to 0.53 at an average of 0.31. HRM has been demonstrated to be a fast, low cost, and efficient approach for SNP discovery and genotyping, in particular, for species without much genomic information such as almond.     

High‐resolution melt analysis without DNA extraction affords rapid genotype resolution and species identification

Extracting and sequencing DNA from specimens can impose major time and monetary costs to studies requiring genotyping, or identification to species, of large numbers of individuals. As such, so‐called direct PCR methods have been developed enabling significant savings at the DNA extraction step. Similarly, real‐time quantitative PCR techniques (qPCR) offer very cost‐effective alternatives to sequencing. High‐resolution melt analysis (HRM) is a qPCR method that incorporates an intercalating dye into a double‐stranded PCR amplicon. The dye fluoresces brightly, but only when it is bound. Thus, after PCR, raising the temperature of the amplicon while measuring the fluorescence of the reaction results in the generation of a sequence‐specific melt curve, allowing discrimination of genotypes. Methods combining HRM (or other qPCR methods) and direct PCR have not previously been reported, most likely due to concerns that any tissue in the reaction tube would interfere with detection of the fluorescent signal. Here, we couple direct PCR with HRM and, by way of three examples, demonstrate a very quick and cost‐effective method for genotyping large numbers of specimens, using Rotor‐Gene HRM instruments (QIAGEN). In contrast to the heated‐block design of most qPCR/HRM instruments, the Rotor‐Gene's centrifugal rotor and air‐based temperature‐regulation system facilitate our method by depositing tissues away from the pathway of the machine's fluorescence detection optics.     

High-Resolution DNA Melt Curve Analysis of the Clustered, Regularly Interspaced Short-Palindromic-Repeat Locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.     

High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni

A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.     

人-牛异种克隆胚构建及其线粒体来源

通过人-牛异种核移植技术获得异种克隆囊胚, 便于在不消耗人类卵母细胞的情况下从异种克隆胚中分离出人类干细胞。通过透明带下注射法将人胎儿成纤维细胞和牛耳成纤维细胞分别注入去核牛卵母细胞中构建异种和同种胚胎, 并比较两者之间的融合率、卵裂率、8-细胞发育率以及囊胚率。并对处于2-细胞、4-细胞、8-细胞、桑椹胚、囊胚阶段的异种克隆胚的线粒体DNA来源进行检测。结果表明, 异种克隆胚体外各个阶段的发育率均低于同种克隆胚, 尤其是8-细胞到囊胚阶段的发育率, 以及囊胚率都显著低于同种克隆胚(P<0.05)。异种克隆胚在2-细胞到桑椹胚阶段检测到人、牛线粒体DNA共存, 囊胚阶段只检测到牛线粒体DNA。结果表明: 牛卵母细胞可以重编程人胎儿成纤维细胞, 完成异种克隆胚植入前的胚胎发育, 异种克隆胚由于核质相互作用的不谐调, 影响其发育能力, 使其囊胚率显著低于同种克隆胚。牛线粒体DNA存在于植入前异种胚胎发育的各个阶段。异种克隆胚胎用于人类胚胎干细胞分离具有可行性。     

An efficient method for screening faecal DNA genotypes and detecting new individuals and hybrids in the red wolf (<Emphasis Type="Italic">Canis rufus</Emphasis>) experimental population area

Previously, sequencing of mitochondrial DNA (mtDNA) from non-invasively collected faecal material (scat) has been used to help manage hybridization in the wild red wolf (Canis rufus) population. This method is limited by the maternal inheritance of mtDNA and the inability to obtain individual identification. Here, we optimize the use of nuclear DNA microsatellite markers on red wolf scat DNA to distinguish between individuals and detect hybrids. We develop a data filtering method in which scat genotypes are compared to known blood genotypes to reduce the number of PCR amplifications needed. We apply our data filtering method and the more conservative maximum likelihood ratio method (MLR) of Miller et al. (2002 Genetics 160:357–366) to a scat dataset previously screened for hybrids by sequencing of mtDNA. Using seven microsatellite loci, we obtained genotypes for 105 scats, which were matched to 17 individuals. The PCR amplification success rate was 50% and genotyping error rates ranged from 6.6% to 52.1% per locus. Our data filtering method produced comparable results to the MLR method, and decreased the time and cost of analysis by 25%. Analysis of this dataset using our data filtering method verified that no hybrid individuals were present in the Alligator River National Wildlife Refuge, North Carolina in 2000. Our results demonstrate that nuclear DNA microsatellite analysis of red wolf scats provides an efficient and accurate approach to screen for new individuals and hybrids.     

Development of a high resolution melting method for genotyping of risk HLA-DQA1 and PLA2R1 alleles and ethnic distribution of these risk alleles

Recent studies have demonstrated that alleles at single nucleotide polymorphisms (SNPs) rs2187668 and rs4664308 within genes HLA-DQA1 and PLA2R1, respectively, had a significant impact on the susceptibility to idiopathic membranous nephropathy (IMN). Analysis of the two genomic loci could identify alleles for individuals at risk for IMN. Conventional methods for genotyping are labor intensive, expensive or time consuming. High resolution melting (HRM) is a new technique for genotyping and has the advantages of simplicity, speed, high sensitivity and low cost. Here, we describe genotyping of SNPs rs2187668 and rs4664308 using HRM. In this study, we identified polymorphisms of rs2187668 and rs4664308 in 480 healthy unrelated Chinese volunteers of two ethnic groups from three different geographical areas in China. The two genomic loci were genotyped by HRM using a saturating fluorescent dye SYTO® 9 on 7900 HT and RG 6000 instruments, and were further confirmed by direct DNA sequencing. Three different SNP genotypes were sufficiently distinguished by HRM with mean sensitivity of 98.8% and mean error rate of 1.9%. In addition, the allele frequencies varied greatly based on ethnic or geographic origins. In conclusion, HRM is a rapid, cost efficient, sensitive, suitable technique for genotyping, and simple enough to be readily implemented in a diagnostic laboratory. We believe this will be a valuable technique for determining the genotype of rs2187668 and rs4664308 and for assessing individual susceptibility to IMN.     

Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population

Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics.     

High-resolution melting analysis to discriminate between the SARS-CoV-2 Omicron variants BA.1 and BA.2

High-resolution melting (HRM) analysis was conducted to discriminate between SARS-CoV-2 Omicron variant BA.1 (B.1.1.529.1) and subvariant BA.2 (B.1.1.529.2). We performed two-step PCR consisting of the first PCR and the second nested PCR to prepare the amplicon for HRM analysis, which detected G339D, N440K, G446S and D796Y variations in the SARS-CoV-2 spike protein. The melting temperatures (Tms) of the amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 receptor binding domain (RBD) in spike protein were the same: 75.2 °C (G339D variation) and 73.4 °C (D796Y variation). These Tms were distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1, and were specific to the Omicron variant. In HRM analyses that detected the N440K and G446S variations, the Tms of amplicons from the cDNA of the Omicron variant BA.1 and subvariant BA.2 RBDs were 73.0 °C (N440K and G446S variations) and 73.5 °C (G446S variation). This difference indicates that the SARS-CoV-2 Omicron variants BA.1 and BA.2 can be clearly discriminated. Our study demonstrates the usefulness of HRM analysis after two-step PCR for the discrimination of SARS-CoV-2 variants.     

Phenotype of FAECB (Facility for Automated Experiments in Cell Biology) Chinese hamster ovary mutants with minimal UV-sensitivity

Mitochondrial DNA (mtDNA) haplogroup U, defined by the polymorphism 12308A>G, may constitute a risk factor for an occipital stroke in migraine. We therefore identified 14 patients with an occipital stroke and with 12308A>G. We determined complete mtDNA coding region sequence for the patients and for population controls by conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Sequence information was used to construct a phylogenetic network of mtDNA haplogroups U and K, which was found to be composed of subclusters U2, U4, U5 and a new subcluster U7, as well as cluster K. Five patients with a migrainous stroke belonged to subcluster U5 (P=0.006; Fisher's exact test). Many unique mutations were found among the patients with an occipital stroke including two tRNA mutations that have previously been suggested to be pathogenic. Analysis of mtDNA sequences by CSGE and comparison of the sequences through phylogenetic analysis greatly enhances the identification of mtDNA clusters in population and detection of mtDNA mutations in patients.     

Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis

Ly49G and H-2 class I D(k) molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I D(k) disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F(2) and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.     

Discrimination of Gastrointestinal Nematode Eggs from Crude Fecal Egg Preparations by Inhibitor-Resistant Conventional and Real-Time PCR

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles – only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can be used such as RFLP, reverse-line-blot, Sanger sequencing and HRM.     

A novel genotyping method for detection of the CRHR1 (rs1396862: C>T) gene variation among North Indian population

Today, the genomic revolution in epidemiology, medicine and population based genetic association studies are results of on-going refocusing efforts toward development of inexpensive and accurate techniques for SNP genotyping. Despite this considerable gain, high throughput and routinely applicable newer SNP detection techniques are still needed. Therefore, aim of this study was to develop and validate a simple, rapid and inexpensive restriction enzyme based method for genotyping of corticotrophin-releasing hormone receptor1 (CRHR1; rs1396862: C>T) gene variant. This polymorphism has been investigated in a variety of psychiatric and association studies of asthma. A total of 250 healthy volunteers were recruited from same ethnicity and their blood DNA samples were employed for genotyping. Primers were designed using Batch primer3 Software. Specificity and functionality of primers were tested with BLAST database and UCSC In-silco PCR respectively. The lake of a PstI recognition site was seen with T allele. The allele frequencies for rs1396862: C>T were 0.88 (C allele) and 0.12 (T allele). We get 100 % concordant genotyping results for sequencing and PCR–RFLP. This newer genotyping approach lowers the cost and increased the speed. It is particularly useful for small basic research studies of complex genetic disorder.     

Genotyping on ALDH2: Comparison of Four Different Technologies

Objectives

This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and Methods

We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results

All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions

All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.     

Application of high-resolution DNA melting for genotyping in lepidopteran non-model species: Ostrinia furnacalis (Crambidae)

Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.     

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.     

In silico single nucleotide polymorphism discovery and application to marker-assisted selection in soybean

The general approach to discovering single nucleotide polymorphisms (SNPs) requires locus-specific PCR amplification. To enhance the efficiency of SNP discovery in soybean, we used in silico analysis prior to re-sequencing as it is both rapid and inexpensive. In silico analysis was performed to detect putative SNPs in expressed sequence tag (EST) contigs assembled using publicly available ESTs from 18 different soybean genotypes. SNP validation by direct sequencing of six soybean cultivars and a wild soybean genotype was performed with PCR primers designed from EST contigs aligned with at least 5 out of 18 soybean genotypes. The efficiency of SNP discovery among the confirmation genotypes was 81.2%. Furthermore, the efficiency of SNP discovery between Pureunkong and Jinpumkong 2 genotypes was 47.4%, a great improvement on our previous finding based on direct sequencing (22.3%). Using SNPs between Pureunkong and Jinpumkong 2 in EST contigs, which were linked to target traits, we were able to genotype 90 recombinant inbred lines by high-resolution melting (HRM) analysis. These SNPs were mapped onto the expected locations near quantitative trait loci for water-logging tolerance and seed pectin concentration. Thus, our protocol for HRM analysis can be applied successfully not only to genetic diversity studies, but also to marker-assisted selection (MAS). Our study suggests that a combination of in silico analysis and HRM can reduce the cost and labor involved in developing SNP markers and genotyping SNPs. The markers developed in this study can also easily be applied to MAS if the markers are associated with the target traits.     

The utility of high-resolution melting analysis of SNP nucleated PCR amplicons--an MLST based Staphylococcus aureus typing scheme

High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G?A, G?T, C?A, C?T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 "melt types" (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs.