Mutation Detection by Fluorescent Hybridization Probe Melting Curves

2001 ◽  
pp. 11-19 ◽  
Author(s):  
Philip S. Bernard ◽  
Astrid Reiser ◽  
Gregory H. Pritham
Keyword(s):  
Mutation Detection ◽  
Hybridization Probe ◽  
Melting Curves

scholarly journals Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V with a Hybridization Probe Genotyping Assay on the LightCycler

1999 ◽  
Vol 45 (12) ◽  
pp. 2094-2101 ◽  
Author(s):  
Nicolas von Ahsen ◽  
Michael Oellerich ◽  
Victor William Armstrong ◽  
Ekkehard Schütz
Keyword(s):  
Nucleic Acid ◽  
Apolipoprotein B ◽  
Mutation Detection ◽  
Nearest Neighbor ◽  
Molecular Diagnostic ◽  
Probe Design ◽  
Factor V ◽  
Melting Points ◽  
Hybridization Probe ◽  
Genotyping Assay

Abstract Background: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations. Methods: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented. Results: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; Sy|x= 0.96; n = 24). Conclusions: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.

Mutation Detection of K-ras Gene in Paraffin-embedded Colorectal Cancer Tissues by Using Chip-based TGCE*

2010 ◽  
Vol 37 (7) ◽  
pp. 794-800 ◽  
Author(s):  
Hui-Dan ZHANG ◽  
Xiao-Nan WANG ◽  
Zhe ZHOU ◽  
Qian MA ◽  
Jin FANG
Keyword(s):  
Colorectal Cancer ◽  
Mutation Detection ◽  
Cancer Tissues ◽  
I Gene

scholarly journals Retinoblastoma genetics screening and clinical management

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Himika Gupta ◽  
Sivasankar Malaichamy ◽  
Ashwin Mallipatna ◽  
Sakthivel Murugan ◽  
Nallathambi Jeyabalan ◽  
...  
Keyword(s):  
Clinical Management ◽  
Detection Rate ◽  
Mutation Detection ◽  
Gene Mutations ◽  
Disease Recurrence ◽  
Germline Mutations ◽  
Global Studies ◽  
Mutation Detection Rate ◽  
Gene Deletions

Abstract Background India accounts for 20% of the global retinoblastoma (RB) burden. However, the existing data on RB1 gene germline mutations and its influence on clinical decisions is minimally explored. Methods Fifty children with RB underwent complete clinical examination and appropriate multidisciplinary management. Screening of germline RB1 gene mutations was performed through next-generation sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. The mutation and non-mutation groups were compared for clinical parameters especially severity, progression and recurrence. Results Twenty-nine patients had bilateral RB (BLRB) and 21 had unilateral RB (ULRB). The genetic analysis revealed 20 RB1 variations in 29 probands, inclusive of 3 novel mutations, known 16 mutations and heterozygous whole gene deletions. The mutation detection rate (MDR) was 86.2% in BLRB and 19% in ULRB. Associations of disease recurrence (p = 0.021), progression (p = 0.000) and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors were observed in the mutation group. Clinical management was influenced by the presence of germline mutations, particularly while deciding on enucleation, frequency of periodic follow up and radiotherapy. Conclusions We identified novel RB1 mutations, and our mutation detection rate was on par with the previous global studies. In our study, genetic results influenced clinical management and we suggest that it should be an essential and integral component of RB-care in India and elsewhere.

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