High resolution melting analysis for gene scanning

High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the focus of this article. With the simple addition of a heteroduplex-detecting dye before PCR, high resolution melting is performed without any additions, processing or separation steps. Heterozygous variants are identified by atypical melting curves of a different shape compared to wild-type homozygotes. Homozygous or hemizygous variants are detected by prior mixing with wild-type DNA. Design, optimization, and performance considerations for high resolution scanning assays are presented for rapid turnaround of gene scanning. Design concerns include primer selection and predicting melting profiles in silico. Optimization includes temperature gradient selection of the annealing temperature, random population screening for common variants, and batch preparation of primer plates with robotically deposited and dried primer pairs. Performance includes rapid DNA preparation, PCR, and scanning by high resolution melting that require, in total, only 3 h when no variants are present. When variants are detected, they can be identified in an additional 3 h by rapid cycle sequencing and capillary electrophoresis. For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.     

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3′-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.     

Rapid genotyping of APOA5 -1131T>C polymorphism using high resolution melting analysis with unlabeled probes

The APOA5 -1131 T/C polymorphism (rs662799) exhibits a very strong association with elevated TG levels in different racial groups. High resolution melting (HRM) analysis with the use of unlabeled probes has shown to be a convenient and reliable tool to genotyping, but not yet been used for detecting rs662799 polymorphism. We applied the unlabeled probe HRM analysis and direct DNA sequencing to assay the -1131T>C SNP in 130 cases DNA samples blindly. This HRM analysis can be completed in <3 min for each sample. The two melting peaks were displayed at 66.1±0.4°C for CC homozygote and 68.7±0.2°C for TT homozygote; TC heterozygote showed the both melting peaks. The genotyping results by HRM method were completely concordant with direct DNA sequencing. The distribution of CC, TC, and TT genotypes for the -1131T>C SNP was 9.2, 49.2, and 41.5%, respectively. This assay was sensitive enough to detect C allele down to 20% and 10% for T allele. The limit of detection for C and T allele was 6.2 and 2.5 ng/μL DNA, respectively. The developed unlabeled probe HRM method provides an alternative mean to detect ApoA5 -1131T>C SNP rapidly and accurately.     

Automated DNA Extraction,Quantification, Dilution,and PCR Preparation for Genotyping by High-Resolution Melting

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T_m). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T_m, as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.     

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the “closed tube” format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the “open tube” format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5–SYBR Green I and Bio-Rad CFX96–EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a “lower level” into the range of those suitable for mutation scanning.     

Scanning of mutations in short amplicons: Optimization of DNA melting method

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (postamplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the open-tube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to “Sanger” sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.     

Application of high-resolution melting for genotyping bovine mitochondrial DNA

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions.     

Optimization of melting analysis with TaqMan probes for detection of KRAS,NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔT_m (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.     

Multiplex genotyping by melting analysis of loci-spanning probes: beta-globin as an example

Multiplexing genotyping technologies usually require as many probes as genetic variants. Oligonucleotides that span multiple loci--loci spanning probes (LSProbes)--hybridize to two or more noncontiguous DNA sequences present in a template and can be used to analyze multiple variants simultaneously. The intervening template sequence, omitted in the LSProbe, creates a bulge-loop during binding. Melting temperatures of the probe, monitored by fluorescence reading are specific to the presence or absence of the mutations. We previously described LSProbes as a molecular haplotyping tool and apply here the principle to genotype simultaneously three mutations of the beta-globin gene responsible for the corresponding hemoglobinopathies. Analysis with both labeled and unlabeled LSProbes demonstrate that the four possible alleles studied (WT, HbS, HbC, and HbE) are identifiable by the specific melting temperatures of the LSProbes. This demonstrates that, in addition to their haplotyping capabilities, LSProbes are able to genotype in a single step, loci 58 nucleotides apart.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

高分辨率熔解曲线法的非标记探针基因分型技术

PCR反应中利用荧光检测技术对已知位点进行基因分型时常采用荧光标记的寡核苷酸做探针。近年来新兴起的高分辨率熔解曲线技术可以采用非标记的探针对已知位点的SNP(single nucleotide polymorphism)或突变进行基因分型研究。采用非标记探针法对已知位点的基因分型研究具有廉价、快速、简便等特点,因此被大量应用在和疾病、形状等相关的一些多肽位点的研究中。本文较详细地介绍该技术的基本原理和实验中的注意事项。     

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.     

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR Gene ImagIR2 DNA analyzer

Inter-simple sequence repeat (inter-SSR) PCR was assessed for use in variety testing of chrysanthemum. This method was modified to allow detailed analysis of DNA profiles on a LI-COR Gene ImagIR2 DNA analyzer system. Protocols for unlabeled PCR were unsuccessful in producing labeled products when using infrared (IR) dye-labeled primers. Various modifications to the known protocols were investigated: (i) different ratios of labeled to unlabeled primer; (ii) various annealing temperatures; (iii) the use of an IR genotyping kit; (iv) end labeling; and (v) direct incorporation and cycle labeling. Successful amplification using labeled primers only occurred when two consecutive reactions were performed. The first PCR was performed using standard protocols for unlabeled reactions. The second PCR used a dilution of these reaction products as a template and 50% IR-labeled and unlabeled primer. The complete procedure leading to a high-resolution analysis of inter-SSR PCR products on a LI-COR system is reported for the first time. This system allows high-throughput fingerprinting with the potential for applications on a commercial scale.     

Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.     

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

Pyrosequencing protocol using a universal biotinylated primer for mutation detection and SNP genotyping

DNA sequencing has markedly changed the nature of biomedical research, identifying millions of polymorphisms along the human genome that now require further analysis to study the genetic basis of human diseases. Among the DNA-sequencing platforms available, Pyrosequencing has become a useful tool for medium-throughput single nucleotide polymorphism (SNP) genotyping, mutation detection, copy-number studies and DNA methylation analysis. Its 96-well genotyping format allows reliable results to be obtained at reasonable costs in a few minutes. However, a specific biotinylated primer is usually required for each SNP under study to allow the capture of single-stranded DNA template for the Pyrosequencing assay. Here, we present an alternative to the standard labeling of PCR products for analysis by Pyrosequencing that circumvents the requirement of specific biotinylated primers for each SNP of interest. This protocol uses a single biotinylated primer that is simultaneously incorporated into all M13-tagged PCR products during the amplification reaction. The protocol covers all steps from the PCR amplification and capture of single-stranded template, its preparation, and the Pyrosequencing assay itself. Once the correct primer stoichiometry has been determined, the assay takes around 2 h for PCR amplification, followed by 15-20 min (per plate) to obtain the genotypes.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.