Detection Of Single Base Substitutions Research Articles

DCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

Abstract 1665: Development of standardized assay and software systems to deliver concordant results across sequencing platforms

Abstract Introduction The role of NGS testing to inform clinical decisions has greatly expanded, now including biomarkers such as microsatellite instability (MSI) and Tumor Mutational Burden (TMB) for immune therapies. As oncology adopts widespread clinical use of NGS, standardization becomes essential. PGDx is developing IVD NGS assays optimized for these complex and comprehensive tumor profiling applications. Here, we describe the analytical performance for detection of various analytes using the PGDx elioTM tissue hybrid capture-based assay on the Ion Torrent S5 NGS platform. Notably, standardized and systematic design of wet chemistry and bioinformatics algorithms in combination yields high-performing results, regardless of sequencing platforms. Method The comprehensive PGDx assay covers the coding regions of >500 genes, complex structural alterations, and genomic signatures associated with solid cancers. Specifically, single-base substitutions (SBS), amplifications, fusions, MSI and TMB are analyzed. A custom bioinformatics pipeline was developed to accommodate single-end Ion Torrent reads with analyte-specific algorithms. PGDx’s VariantDx software was used to detect variants with allele frequencies > 2%. MSI, amplifications and rearrangements were identified using proprietary methods and algorithms. TMB was calculated from non-synonymous somatic variants across the panel, excluding common germline alterations. TMB was normalized to the targeted region to achieve a mutation density of mutations per megabase. To determine analytical accuracy, we compared TMB values from the PGDx assay against a validated whole-exome sequencing (WES) method for 93 lung cancer patients. Results TMB results demonstrated a Spearman correlation coefficient of >0.90 compared to whole-exome TMB. MSI and amplifications were detected with 100% analytical accuracy, compared to orthogonal Illumina-based methods (n=19). SBS achieved >94% agreement compared to samples sequenced with Illumina-based methods, covering the same >500 gene panel. ROS1 and RET rearrangements were detected in 100% of 20 cell-line replicates obtained from Horizon®. Conclusion Collectively, these data indicate the systematic co-development of a hybrid capture-based NGS assay with bioinformatics software is practical on the Ion Torrent S5 NGS platform. PGDx elio comprehensive tissue assays may be utilized for detection of SBSs, amplifications, fusions, MSI and TMB on the Ion Torrent S5 NGS platform with robust correlation to Illumina-based results and gold-standard WES. Citation Format: Sudhir Chowbina, Gustavo Cerqueira, Deacon Sweeney, Shantanu Shewale, James Hernandez, Eniko Papp, Mark Sausen, John Thompson, David Riley, Sam Angiuoli. Development of standardized assay and software systems to deliver concordant results across sequencing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1665.

A model-based analysis method for detection of single-base substitution using resequencing microarrays

A model-based analysis method for detection of single-base substitution using resequencing microarrays

Nature and mRNA effect of 282 differentNF1point mutations: focus on splicing alterations

Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutations in the NF1 gene. In this paper we report our experience using the cDNA-SSCP/HD analysis as a mutational screening approach and the double characterization of all mutations at the DNA and RNA levels. Two hundred and eighty-two different mutations (in 374 independent patients) were identified, 140 of which were novel in our population. Most of these mutations are unique and distributed along the gene. However, we also detected 37 recurrent mutations. Our approach is limited with respect to the detection of single base substitutions, but it is highly effective in the detection of frameshift mutations and mutations that affect the correct splicing. Due to this bias we focus here in the characterization of these two types of mutations. Forty-seven percent of mutations found were frameshift mutations, with small deletions being 2.3 times more common than small insertions. At the mRNA level, 44% of mutations affected the correct splicing, 80% of them located in the consensus sequences, with the donor site being much more frequently involved. The remaining 20% consisted of mutations located in deep intronic sites and mutations located in the coding region. In general the latter group produces different types of mutated transcripts with specific proportions for each mutation. The double characterization of mutations at the DNA and RNA levels enables to detect a broader spectrum of mutations than any single level approach, and provides a greater understanding of their molecular pathogenesis.

DNA Diagnostics by Surface-Bound Melt-Curve Reactions

Melting-curve procedures track DNA denaturation in real time and so provide an effective way of assessing sequence variants. Dynamic allele-specific hybridization (DASH) is one such method, based on fluorescence, which uses heat to denature a single allele-specific probe away from one strand of a polymerase chain reaction product attached to a solid support. DASH is a proven system for research genotyping, but here we evaluate it for DNA diagnostics under two scenarios. First, for mutation scanning (resequencing), a human genomic sequence of 97 bp was interrogated with 15 probes tiled with 12-base overlaps, providing up to fourfold redundancy per base. This test sequence spanned three high-frequency single nucleotide polymorphisms, all of which were correctly revealed in 16 individuals. Second, to score multiple different mutations in parallel, 18 alterations in the gyrA gene of Salmonella were assessed in 62 strains by using wild-type- and mutation-specific probes. Both experiments were performed in a blinded manner, and all results were confirmed by sequencing. All DNA variants were unambiguously resolved in every sample, with no false-positive or false-negative signals across all of the investigations. In conclusion, DASH performs accurately and robustly when applied to DNA diagnostic challenges, including mutation scoring and mutation scanning.

Detection of Single-Base Substitutions in Amplified Fragments via Ligation of a Tandem of Short Oligonucleotides in Solution and on a Solid Carrier

Ligation of a tandem of short oligonucleotides was proposed for detecting single-base substitutions in amplified DNA fragments. An octamer–tetramer–octamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligation did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the octamer immobilized on methacrylate beads (DMEG). In both cases, polymer attachment improved the discrimination of 20-mer templates with single-base substitutions in the binding site for the tetramer or for the immobilized octamer. Tandems with a radiolabeled or biotinylated component were also efficiently ligated on amplified DNA fragments. The data obtained with DNA fragments of HIV-1 strains bru and rf demonstrate the possibility of reliable detection of single-base substitutions via ligation of a tandem and colorimetric detection of the immobilized ligation product with the streptavidin–alkaline phosphatase technique.

Allele-specific hybridization of lipoprotein lipase and factor-V Leiden missense mutations with direct label alkaline phosphatase-conjugated oligonucleotide probes

Direct label alkaline phosphatase (AP) conjugated oligonucleotide probes (AP-DNA) were prepared to assess their utility for allele-specific detection of single base substitutions. Oligonucleotide conjugates were designed to detect point mutations in the genes for lipoprotein lipase (LPL) and coagulation factor-V (FV). Genomic DNA samples, including ones known to harbor point mutations in the genes for LPL and FV, were prepared from whole blood and subjected to polymerase chain reaction (PCR). PCR products were analyzed by Southern hybridization with the allele-specific AP-DNA probes and restriction endonuclease analysis. Thermal profiles for hybridization indicate optimal allele-specific selectivity was achieved with temperatures ranging from 45°C to 55°C at a total Na + concentration of 150 mM. Under these conditions the base changes studied were easily discriminated with allele specific hybridization signals in excess of 200:1 as estimated by scanning densitometry. Complete concordance was observed between hybridization and restriction analyses for 175 LPL and 201 FV clinical and reference samples. The total time for analysis of the PCR products was less than 2 h with a dot blot hybridization protocol.

Analysis of polymerase chain reaction product by capillary electrophoresis and its application to the detection of single base substitution in genes

Capillary gel electrophoresis (CGE) was studied for the direct analysis of polymerase chain reaction (PCR) amplified samples. A low cross-linked polyacrylamide gel (3%T, 0.5%C) was used for CGE with treated and untreated silica capillaries. CGE showed high reproducibility and resolution in the separation of DNA fragments ( ca. 100–1000 base pairs) produced by PCR. The CGE system was applied to the detection of an amplification refractory mutation system (ARMS) and PCR—restriction fragment length polymorphism (PCR-RFLP), which are detection methods of single base substitution in genes using PCR. With the CGE system, full automation of PCR product detection is feasible.

A simple method for detecting single base substitutions and its application to HLA-DPB1 typing.

We have developed a simple and reliable method, PCR-PHFA (polymerase chain reaction dependent preferential homoduplex formation assay), for detection of single base substitutions within PCR amplicons. This technique is based upon strand competition during hybridization between a double labeled amplicon, prepared from biotin and DNP labeled primers, and an unlabeled amplicon. Under the precisely controlled temperature gradient, the preferential formation of a homoduplex over a heteroduplex occurs. After annealing, the identical sequence of the double labeled and unlabeled amplicon resulted in a low population of regenerated double labeled dsDNA due to strand exchange between them. Even when the two differed by only a single base substitution, double labeled molecule was regenerated efficiently because of preferential homoduplex formation. The regenerated double labeled molecule was captured onto a streptavidin coated microtiter plate and quantified enzymatically with a chromogenic substrate. The technique has been successfully applied in HLA-DPB1 typing. Furthermore, we detected a mutated gene even in the presence of a large excess of the corresponding normal gene.

Detection of single base substitutions in polynucleotides by capture with immobilized oligonucleotides

We have improved a sandwich hybridization assay to detect single base substitutions in polymerase chain reaction (PCR) amplified DNA sequences. The target DNA was captured by an immobilized oligonucleotide and revealed using a second oligonucleotide coupled to an enzyme. Short oligonucleotides (13, 15 bases) were used to obtain specific hybridization at 37°C. We developed two different assay formats for rapid identification of PCR products: a microtitration plate format with oligonucleotides bound to polystyrene and a channelling assay using oligonucleotides immobilized on Sepharose, which did not require any separation step. The specificity and advantages of both methods are described.

Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations.

The efficiency of detection of single base substitutions by single-stranded conformation polymorphism (SSCP) analysis was tested on 86 randomly distributed point mutations in a 193-bp-long DNA fragment of the mouse beta-globin gene. Multiple parameters were varied, including electrophoresis temperature, buffer concentration, gel concentration, acrylamide-to-bis-acrylamide ratio, and/or addition of different compounds to the gel matrix. Gels with a higher concentration of acrylamide and lower crosslinking gave optimal separation, and all 86 mutations can be clearly distinguished from the wild type on a 5% or 7.5% (2.6% C) acrylamide gel at 4 degrees C. Most of the mutations are also resolvable from wild type on gels with 5% urea or formamide, or 10% dimethylsulfoxide or sucrose. The relative position of the purine and pyrimidine-rich single strands were followed by an asymmetric PCR-SSCP technique. We found that most of the informativity comes from the purine rich strand, which appears to be much more sensitive to changes in the gel. The position or type of mutation showed no correlation with its ability to be detected. However, the neighboring base sequence around the mutation appears to have an effect on mobility. For example, A-->G substitutions in GC-rich regions significantly increase the mobility shift of the purine-rich strand, while most G-->A changes decrease it. We conclude that SSCP is a very efficient method for the detection of point mutations, if the parameters that effect the separation are optimized for a particular DNA fragment.

The ligase chain reaction in a PCR world.

S i n c e its discovery in 1985, the polymerase chain reaction (PCR) has had a profound impact on detecting genetic and infectious diseases, identifying new genes, and unraveling the mysteries of protein-ligand recognition.(1-s) Its universal utility is due to the exquisite specificity of amplification and ease of cycling made possible by the cloning and careful characterization of a thermostable polymerase from Thermus aquaticus. (6,7) Likewise, cloning of a thermostable ligase enabled a new amplification method, termed ligase chain reaction (LCR), to both amplify DNA and discriminate a single base mutation. (8-1~ Although these DNA amplification techniques are new, they bring to fruition the enzymes as reagents philosophy expounded by A. Kornberg and I.R. Lehman a quarter of a century ago. Allele-specific LCR employs four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand (see Fig. 1, top left). Thermostable DNA ligase will covalently link each set, provided that there is complete complementarity at the junc~ tion. (8) Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to PCR amplification. A single-base mismatch at the oligonucleotide junction will not be amplified and is therefore distinguished (Fig. 1, top right). A second set of mutantspecific oligonucleotides is used in a separate reaction to detect the mutant allele. This review will give a brief introduction to (1) determinants of specificity in amplification reactions; (2) differences between thermostable and thermophilic enzymes; (3) detection of single-base substitutions in DNA diagnostics; (4) characterization of DNA ligases; (5) cloning of DNA ligases; (6) use of ligases in DNA detection; (7) other methods that use ligase, such as ligase-mediated PCR and the branch capture reaction; and (8) potential new uses of ligase in PLCR and nested LCR amplification reactions. It will close with speculations on future prospects.

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.

Detection of single base substitutions in influenza virus RNA molecules by denaturing gradient gel electrophoresis of RNA-RNA or DNA-RNA heteroduplexes

Single point mutations in the NS gene of influenza virus were detected by electrophoresis of double-stranded RNA heteroduplexes in denaturing gradient gels. The heteroduplex RNAs were made by hybridization of virion RNA with SP6-derived RNA probes of varying length. Mutations located at different positions along the NS gene (890 nucleotides long) were all detected in a predictable fashion. The method of heteroduplex analysis was also successfully used in detecting single point mismatches in DNA-RNA hybrids.

Detection of single base substitutions in total genomic DNA.

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.