Allele-specific PCR analysis for detection of the gld Fas-ligand point mutation

1997 ◽  
Vol 210 (1) ◽  
pp. 109-112 ◽  
Author(s):  
Robert M Hoek ◽  
Marion C Kortekaas ◽  
Jonathan D Sedgwick
Keyword(s):  
Point Mutation ◽  
Fas Ligand ◽  
Pcr Analysis ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

2017 ◽  
Author(s):  
Sergey V. Prykhozhij ◽  
Charlotte Fuller ◽  
Shelby L. Steele ◽  
Chansey J. Veinotte ◽  
Babak Razaghi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Point Mutations ◽  
Mutation Site ◽  
Transmission Rates ◽  
Specific Pcr ◽  
Allele Specific ◽  
Pcr Assays ◽  
Combination Of Methods ◽  
Mutation Strategies ◽  
Allele Specific Pcr

AbstractSingle-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

scholarly journals Identification of Self-incompatibility Genotypes in Apple and Crabapple Cultivars by S-allele Specific PCR Analysis

2014 ◽  
Vol 46 (4) ◽  
pp. 364-371
Author(s):  
Kang Hee Cho ◽  
Jeong-Hee Kim2 ◽  
Jung Woo Lee ◽  
Soon-Il Kwon ◽  
Jong Taek Park ◽  
...  
Keyword(s):  
Pcr Analysis ◽  
Self Incompatibility ◽  
Specific Pcr ◽  
Allele Specific ◽  
S Allele ◽  
Allele Specific Pcr

scholarly journals Allele-specific PCR analysis ofp53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis

1996 ◽  
Vol 68 (1) ◽  
pp. 21-25 ◽  
Author(s):  
Gordon M. Kirby ◽  
Gerald Batist ◽  
Nasser Fotouhi-Ardakani ◽  
Hisayoshi Nakazawa ◽  
Hiroshi Yamasaki ◽  
...  
Keyword(s):  
Viral Hepatitis ◽  
Pcr Analysis ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Liver Tissues

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

2000 ◽  
Vol 101 (3) ◽  
pp. 344-349 ◽  
Author(s):  
M. Tamura ◽  
K. Ushijima ◽  
H. Sassa ◽  
H. Hirano ◽  
R. Tao ◽  
...  
Keyword(s):  
Pcr Analysis ◽  
Self Incompatibility ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

scholarly journals Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhengjie Chen ◽  
Dengguo Tang ◽  
Jixing Ni ◽  
Peng Li ◽  
Le Wang ◽  
...  
Keyword(s):  
Marker Assisted Selection ◽  
Inbred Lines ◽  
Average Density ◽  
Snp Markers ◽  
Data Sets ◽  
Rna Seq ◽  
Specific Pcr ◽  
Maize Inbred Lines ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

scholarly journals Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 982
Author(s):  
Zhiliang Xiao ◽  
Congcong Kong ◽  
Fengqing Han ◽  
Limei Yang ◽  
Mu Zhuang ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Brassica Oleracea ◽  
Rapid Identification ◽  
Inbred Lines ◽  
Hybrid Lethality ◽  
Specific Pcr ◽  
Allele Specific ◽  
User Friendly ◽  
Allele Specific Pcr ◽  
Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

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