Accurate Single-Nucleotide Polymorphism Allele Assignment in Trisomic or Duplicated Regions by Using a Single Base–Extension Assay with MALDI-TOF Mass Spectrometry

Trewick, Anne L.; Moustafa, Julia S. El-Sayed; Smith, Adam J. de; Froguel, Philippe; Greve, Gottfried; Njølstad, Pål R.; Coin, Lachlan; Blakemore, Alexandra I. F.

doi:10.1373/clinchem.2010.159558

Cited by 9 publications

(4 citation statements)

References 14 publications

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“…SS genotyping can be used to distinguish between single nucleotide variants in all sequence contexts and does not require reagents or effort beyond PCR. Further, unlike single-base extension genotyping ( Sauer et al 2000 ; Trewick et al 2011 ), the 5′ fluorogenic nuclease Taqman assay ( Livak et al 1995 ; Callegaro et al 2006 ), and melting curve analysis of FRET probes ( Livak 1999 ; Combrinck et al 2013 ), expensive equipment and specialized training are not required for design and use of SS primers for allele detection.…”

Section: Discussionmentioning

confidence: 99%

A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

Touroutine¹,

Tanis²

2020

Genetics

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With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in Caenorhabditiselegans. Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a non-complementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.

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Section: Discussionmentioning

confidence: 99%

A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

Touroutine¹,

Tanis²

2020

Genetics

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show abstract

“…SS genotyping can be used to distinguish between mismatches in all genetic contexts and does not require any reagents or effort beyond PCR. Further, unlike single-base extension genotyping (Sauer 2000; Trewick et al . 2011), the 5’ fluorogenic nuclease Taqman assay (Livak et al .…”

Section: Discussionmentioning

confidence: 99%

A rapid, super-selective method for detection of single nucleotide variants inC. elegans

Touroutine

Tanis

2020

Preprint

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61With the widespread use of single nucleotide variants generated through mutagenesis 62 screens, the million mutation project, and genome editing technologies, there is 63 pressing need for an efficient and low-cost strategy to genotype single nucleotide 64 substitutions. We have developed a rapid and inexpensive method for detection of point 65 mutants through optimization of SuperSelective (SS) primers for end point PCR in 66 Caenorhabditis elegans. Each SS primer consists of a 5' "anchor" that hybridizes to the 67 template, followed by a non-complementary "bridge," and a "foot" corresponding to the 68 target allele. The foot sequence is short, such that a single mismatch at the terminal 3' 69 nucleotide destabilizes primer binding and prevents extension, enabling discrimination 70 of different alleles. We explored how length, stability, and sequence composition of 71 each SS primer segment affected selectivity and efficiency in order to develop simple 72 rules for primer design that allow for distinction between any mismatches in various 73 genetic contexts over a broad range of annealing temperatures. Manipulating bridge 74 length affects amplification efficiency, while modifying the foot sequence can increase 75 discriminatory power. Flexibility in the positioning of the anchor enables SS primers to 76 be used for genotyping in regions with sequences that are challenging for standard 77 primer design. In summary, we have demonstrated flexibility in design of SS primers 78 and their utility for genotyping in C. elegans. Since SS primers reliably detect single 79 nucleotide variants, we propose that this method could have broad application for SNP 80 mapping, screening of CRISPR mutants, and colony PCR to identify successful site-81 directed mutagenesis constructs.82 83 4 84 104 reliable, rapid method for routine genotyping of SNSs.105 5

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“…PLAC4, COL6A2) have been selected for detection. 7,9,20 Moreover, this method could be extended to detect other chromosomal aneuploidies, such as trisomy 13 and trisomy 18 by selecting qualied SNPs on chromosome 13 and chromosome 18 with high heterozygosities.…”

Section: Discussionmentioning

confidence: 99%

“…[6][7][8] However, it was found that the data from MALDI-TOF MS was not so quantitative. 7 Although the quantitative performance of MALDI-TOF MS was improved by using the peak height ratio in MS tracings, 9 unlike ESI-MS, MALDI-TOF MS is still challenging in its quan-tication. In addition, a mass spectrometer used for the assay should be specic to the determination of DNA molecules, for the moment, only iPLEX Gold MALDI-TOF MS system is available in the market, limiting the wide application of this strategy to the clinical screening of Down's syndrome.…”

Section: Introductionmentioning

confidence: 99%

Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR

Huang

et al. 2013

Analyst

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Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quantification performance, pyrosequencing was proposed to replace MS by better quantification of allelic ratios. To achieve a simple and rapid clinical diagnostic, PCR with "HpH Buffer" (a buffer with a high pH) was developed to directly amplify amniotic fluid. By the established assay, 114 samples of amniotic fluid were analyzed by pyrosequencing five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cut-off values for prenatal diagnosis of trisomy 21. The panel of five SNPs were high in heterozygosity so that at least one heterozygote was found in each sample, and 86% of the samples had at least two heterozygotes, giving a nearly 100% sensitivity (population coverage) of the assay. By using the cut-off values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with a confidence level over 99%, indicating that our method and karyotyping analysis were consistent in results. In conclusion, this pyrosequencing-based approach, coupled with direct amplification of amniotic fluid, is accurate in quantitative genotyping and simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.

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Accurate Single-Nucleotide Polymorphism Allele Assignment in Trisomic or Duplicated Regions by Using a Single Base–Extension Assay with MALDI-TOF Mass Spectrometry

Cited by 9 publications

References 14 publications

A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

A rapid, super-selective method for detection of single nucleotide variants inC. elegans

Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR

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