Molecular marker analysis of Cynanchum wilfordii and C. auriculatum using the simple ARMS-PCR method with mismatched primers

Han, Eun-Heui; Lee, Soo Jin; Kim, Man Bae; Shin, Yong-Wook; Kim, Yun-Hee; Lee, Shin-Woo

doi:10.1007/s11816-017-0432-0

Cited by 6 publications

(5 citation statements)

References 13 publications

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“…A large number of molecular marker techniques, such as simple sequence repeat (SSR), single nucleotide polymorphism (SNP), and internal transcribed spacer (ITS), have been used in plant classification, identification, and analysis of genetic characteristics [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. Among them, ITS sequence of nuclear ribosomal DNA (nrDNA) has been proved to be a helpful non-coding marker to infer hybridization events [ 5 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing

et al. 2018

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Codonopsis taxa, as a traditional Chinese medicinal and edible plant, has found expanding domestic and foreign applications in recent decades. However, the poor management in germplasm resources market has inevitably caused an unnecessary hybrid of the provenances. In order to clarify the hybrid characteristics of germplasm resources in the main production area, the Codonopsis cultivars collected from the provinces Gansu, Shannxi, Shanxi, and Hubei of China were researched, using internal transcribed spacer (ITS) sequence technology. The confirmation of additive nucleotides based on the ITS sequencing of polymerase chain reaction (PCR) mixture was optimized and used to study the hybrid of Codonopsis cultivars. The results showed that when the ratio of PCR mixture increased up to 15 percent, the presence of a double peak in the sequencing electrophoresis map could be confirmed, suggesting the existence of additive nucleotides. According to the method above, 46 samples of Codonopsis cultivars collected during 2016 and 2017 were studied and compared with the samples collected from the year 2009 to 2010. All of the samples collected during 2016 and 2017 were hybridized and no genetic pure lines were found. In addition, the sites of variable base reduced greatly, concentrating at positions 122 and/or 226. These phenomena suggested that the genetic diversity of Codonopsis cultivars declined and the germplasm resources gradually converged. More attention should be paid to the reasonable exploitation and genetic breeding of Codonopsis taxa.

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Section: Introductionmentioning

confidence: 99%

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing

et al. 2018

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“…Furthermore, the possibility of artificial mismatches added to the 5′-end of ASP in various methods can be considered (Liu et al, 2012 ; Choi et al, 2017 ; Han et al, 2017 ; Lu et al, 2020 ). Despite some possible increased discrimination between alleles (Hirotsu et al, 2010 ), the artificial mismatches in ASP can cause a reduction of total PCR efficiency, or inhibit the extension of the product by Taq polymerase (Ke and Wartell, 1993 ; Wu et al, 2009 ).…”

Section: Discussionmentioning

confidence: 99%

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

et al. 2022

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The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

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“…This system enables discrimination between the templates with point mutations and normal templates by ensuring that the 3′ end base of the primer specifically complements either the mutation or the wild-type template base. ARMS-PCR can detect single nucleotide mutations and small deletions on DNA molecules simply by introducing mismatched bases in the primer design [20]. This method has wide applications in genetic analysis, mutation identification, and human leukocyte antigen (HLA) typing [21].…”

Section: Introductionmentioning

confidence: 99%

Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples

Tan,

Zhang,

Wei

et al. 2024

CIMB

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A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.

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Molecular marker analysis of Cynanchum wilfordii and C. auriculatum using the simple ARMS-PCR method with mismatched primers

Cited by 6 publications

References 13 publications

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing

Study on Hybrid Characteristics of Medicinally Used Cultivated Codonopsis Species Using Ribosomal Internal Transcribed Spacer (ITS) Sequencing

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples

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