Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems

Abstract Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how “self-avoiding molecular recognition systems” (SAMRS) manage such difficulties. SAMRS nucleobases pair with complementary nucleotides with strengths comparable to the A:T pair, but do not pair with other SAMRS nucleobases. This should allow primers holding SAMRS components to avoid primer–primer interactions, preventing primer dimers, allowing more sensitive SNP detection, and supporting higher levels of multiplex PCR. The experiments here examine the PCR performances of primers containing different numbers of SAMRS components placed strategically at different positions, and put these performances in the context of estimates of SAMRS:standard pairing strengths. The impact of these variables on primer dimer formation, the overall efficiency and sensitivity of SAMRS-based PCR, and the value of SAMRS primers when detecting single nucleotide polymorphisms (SNPs) are also evaluated. With appropriately chosen polymerases, SNP discrimination can be greater than the conventional allele-specific PCR, with the further benefit of avoiding primer dimer artifacts. General rules guiding the design of SAMRS-modified primers are offered to support medical research and clinical diagnostics products.

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Fine Mapping of qd1, a Dominant Gene that Regulates Stem Elongation in Bread Wheat

Frontiers in Genetics ◽

10.3389/fgene.2021.793572 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yongdun Xie ◽

Weiwei Zeng ◽

Chaojie Wang ◽

Daxing Xu ◽

Huijun Guo ◽

...

Keyword(s):

Bread Wheat ◽

Stem Elongation ◽

Dominant Gene ◽

Nucleotide Polymorphisms ◽

Sequencing Data ◽

Specific Pcr ◽

Yield Determination ◽

Allele Specific ◽

Stem Development ◽

Kasp Markers

Stem elongation is a critical phase for yield determination and, as a major trait, is targeted for manipulation for improvement in bread wheat (Triticum aestivum L.). In a previous study, we characterized a mutant showing rapid stem elongation but with no effect on plant height at maturity. The present study aimed to finely map the underlying mutated gene, qd1, in this mutant. By analyzing an F2 segregating population consisting of 606 individuals, we found that the qd1 gene behaved in a dominant manner. Moreover, by using the bulked segregant RNA sequencing (BSR-seq)-based linkage analysis method, we initially mapped the qd1 gene to a 13.55 Mb region on chromosome 4B (from 15.41 to 28.96 Mb). This result was further confirmed in F2 and BC3F2 segregating populations. Furthermore, by using transcriptome sequencing data, we developed 14 Kompetitive Allele-Specific PCR (KASP) markers and then mapped the qd1 gene to a smaller and more precise 5.08 Mb interval from 26.80 to 31.88 Mb. To develop additional markers to finely map the qd1 gene, a total of 4,481 single-nucleotide polymorphisms (SNPs) within the 5.08 Mb interval were screened, and 25 KASP markers were developed based on 10x-depth genome resequencing data from both wild-type (WT) and mutant plants. The qd1 gene was finally mapped to a 1.33 Mb interval from 28.86 to 30.19 Mb on chromosome 4B. Four candidate genes were identified in this region. Among them, the expression pattern of only TraesCS4B02G042300 in the stems was concurrent with the stem development of the mutant and WT. The qd1 gene could be used in conjunction with molecular markers to manipulate stem development in the future.

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Preventing Favism by Selecting Faba Bean Mutants Using Molecular Markers

STEM Fellowship Journal ◽

10.17975/sfj-2017-001 ◽

2017 ◽

Vol 3 (1) ◽

pp. 2-6 ◽

Cited By ~ 1

Author(s):

Melody Song

Keyword(s):

Faba Bean ◽

Expressed Sequence Tag ◽

Nucleotide Polymorphisms ◽

Seed Selection ◽

Single Nucleotide ◽

Specific Pcr ◽

Faba Beans ◽

Allele Specific ◽

Haemolytic Anemia ◽

Allele Specific Pcr

Faba bean (Vicia faba) is an ancient legume species known for its high protein content. The usage and consumption of the faba bean is limited by a glycoside, vicine-convicine (VC). Consumption of VC causes haemolytic anemia in individuals with the genetic condition called favism. Faba beans with low VC concentration are opening the possibility of reduction of favism disease, but there are many challenges in analyzing VC concentration. The objective of this study was to develop expressed sequence tag (EST) markers that can differentiate between low VC content (LVC) and high VC content (HVC) faba bean genotypes. Three single nucleotide polymorphisms (SNPs) were discovered that distinguished between LVC and HVC genotypes. The SNPs were validated using Kompetitive Allele Specific PCR (KASP) and mass spectrometry phenotyping. Molecular marker SNP 316 (Intron of Medtr2g009270 at 1,851,012 bp) was the most successful marker in differentiating between LVC, HVC, and heterozygous faba bean genotypes. This marker has applications in seed selection and acceleration of breeding programs, which is the first step towards allowing all consumers concerned with the effects of favism to enjoy the nutritional value of faba bean.

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TNF-α -308G/A polymorphism and susceptibility to tuberculosis in Azeri population of Iran

Genetika ◽

10.2298/gensr1603819g ◽

2016 ◽

Vol 48 (3) ◽

pp. 819-826 ◽

Cited By ~ 1

Author(s):

Sajjad Ghorghanlu ◽

Mohammad Asgharzadeh ◽

Hossein Samadi-Kafil ◽

Fatemeh Khaki-Khatibi ◽

Jalil Rashedi ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Tuberculosis Patient ◽

P Value ◽

Nucleotide Polymorphisms ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Tuberculosis Patients ◽

Specific Pcr ◽

Allele Specific ◽

And Function

Single nucleotide polymorphisms (SNPs) in cytokine genes may alter the level and function of secreted cytokine; therefore, SNPs can influence the immune response. The aim of the present study was to determine the association of TNF-? -308G/A single nucleotide polymorphism in tuberculosis patients in the Azeri population of Iran. The TNF-308G/A single nucleotide polymorphism in the promoter region was genotyped by using the allele-specific PCR method in 200 healthy controls and 124 tuberculosis patients. The distribution of allele frequencies for TNF-? -308G/A polymorphism between control and tuberculosis patient groups was not significant (P-value = 0.058, OR = 1.5). Furthermore, no statistically significant association was found between TNF-? -308G/A genotype and resistance/susceptibility to TB (P-value = 0.102). Our results suggest that TNF-? -308G/A polymorphism has no measurable effect on the development of tuberculosis in Azeri population of Iran.

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Gold Nanoparticles - based Colorimetric Single Nucleotide Polymorphisms Genotyping Utilizing Allele-specific PCR

IFMBE Proceedings - 5th European Conference of the International Federation for Medical and Biological Engineering ◽

10.1007/978-3-642-23508-5_276 ◽

2011 ◽

pp. 1062-1065

Author(s):

Ye Lim Jung ◽

Cheulhee Jung ◽

Harshala Parab ◽

Hyun Gyu Park

Keyword(s):

Gold Nanoparticles ◽

Single Nucleotide Polymorphisms ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Analysis of Clinically Relevant Single-Nucleotide Polymorphisms by Use of Microelectronic Array Technology

Clinical Chemistry ◽

10.1093/clinchem/48.12.2124 ◽

2002 ◽

Vol 48 (12) ◽

pp. 2124-2130 ◽

Cited By ~ 41

Author(s):

Rosa Santacroce ◽

Antonia Ratti ◽

Francesco Caroli ◽

Barbara Foglieni ◽

Alessandro Ferraris ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Factor Vii ◽

Restriction Enzyme Digestion ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Ret Protooncogene ◽

Mismatched Dna ◽

Specific Pcr ◽

Pcr Products ◽

Allele Specific

Abstract Background: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. Methods: Primer pairs, with one containing a 5′-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, β-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with β-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. Results: Analysis of amplified DNA required 4–6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. Conclusions: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

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Presence of the HPPD Inhibitor Sensitive 1 Gene and ALSS653N Mutation in Weedy Oryza sativa Sensitive to Benzobicyclon

Plants ◽

10.3390/plants9111576 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1576

Author(s):

Chad Brabham ◽

Jason K. Norsworthy ◽

Fidel González-Torralva

Keyword(s):

Weedy Rice ◽

Japonica Rice ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Herbicide Resistant ◽

Rice Plants ◽

Specific Pcr ◽

Kasp Assay ◽

Allele Specific ◽

Allele Specific Pcr

Benzobicyclon has shown varying results in controlling weedy rice, including those with imidazolinone (IMI) resistance. Tolerance to benzobicyclon in cultivated japonica rice, but not indica or aus-like cultivars, is conferred by a fully functional HPPD Inhibitor Sensitive 1 (HIS1) gene. Herein, a diagnostic Kompetitive Allele Specific PCR (KASP) assay was developed to predict the HIS1 genotype of weedy rice plants from 37 accessions and correlated to their response to benzobicyclon in the field. Two-thirds of the 693 weedy rice plants screened were tolerant to benzobicyclon (371 g ai ha−1, SC formulation) at 30 days after treatment (DAT). Thirty-four percent of plants were homozygous for the HIS1 allele and 98% of these plants exhibited field tolerance. However, the his1 genotype did not always correlate with field data. Only 52% of his1 plants were considered sensitive, indicating that the single nucleotide polymorphisms (SNPs) chosen in the KASP assay are not a reliable tool in predicting his1 homozygous plants. In an additional experiment, 86% of the 344 plants with at least one copy of the ALSS653N trait harbored a HIS1 allele, suggesting fields infested with IMI herbicide-resistant weedy rice are unlikely to be controlled with benzobicyclon.

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Single-Tube Genotyping without Oligonucleotide Probes

Genome Research ◽

10.1101/gr.9.1.72 ◽

1999 ◽

Vol 9 (1) ◽

pp. 72-78 ◽

Cited By ~ 2

Author(s):

Søren Germer ◽

Russell Higuchi

Keyword(s):

Pcr Amplification ◽

Oligonucleotide Probes ◽

Nucleotide Polymorphisms ◽

Single Tube ◽

Melting Temperatures ◽

Specific Pcr ◽

Allele Specific ◽

Reverse Primer ◽

Common Reverse Primer ◽

Template Dna

We report the development of a self-contained (homogeneous), single-tube assay for the genotyping of single-nucleotide polymorphisms (SNPs), which does not rely on fluorescent oligonucleotide probes. The method, which we call Tm-shift genotyping, combines allele-specific PCR with the discrimination between amplification products by their melting temperatures (Tm). Two distinct forward primers, each of which contains a 3′-terminal base that corresponds to one of the two SNP allelic variants, are combined with a common reverse primer in a single-tube reaction. A GC-tail is attached to one of the forward allele-specific primers to increase theTm of the amplification product from the corresponding allele. PCR amplification, Tmanalysis, and allele determination of genomic template DNA are carried out on a fluorescence-detecting thermocycler with a dye that fluoresces when bound to dsDNA. We demonstrate the accuracy and reliability ofTm-shift genotyping on 100 samples typed for two SNPs, and recommend it both as a simple and inexpensive diagnostic tool for genotyping medically relevant SNPs and as a high-throughput SNP genotyping method for gene mapping.

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