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Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from...

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Autores principales: Gundry, Cameron N., Dobrowolski, Steven F., Martin, Y. Ranae, Robbins, Thomas C., Nay, Lyle M., Boyd, Nathan, Coyne, Thomas, Wall, Mikeal D., Wittwer, Carl T., Teng, David H.-F.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2425497/
https://www.ncbi.nlm.nih.gov/pubmed/18448472
http://dx.doi.org/10.1093/nar/gkn204
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author Gundry, Cameron N.
Dobrowolski, Steven F.
Martin, Y. Ranae
Robbins, Thomas C.
Nay, Lyle M.
Boyd, Nathan
Coyne, Thomas
Wall, Mikeal D.
Wittwer, Carl T.
Teng, David H.-F.
author_facet Gundry, Cameron N.
Dobrowolski, Steven F.
Martin, Y. Ranae
Robbins, Thomas C.
Nay, Lyle M.
Boyd, Nathan
Coyne, Thomas
Wall, Mikeal D.
Wittwer, Carl T.
Teng, David H.-F.
author_sort Gundry, Cameron N.
collection PubMed
description Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise ∼16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42–86 bp) genotyping on the LightScanner®. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067°C before calibration to 0.022°C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086°C to 0.032°C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical.
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spelling pubmed-24254972008-06-12 Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons Gundry, Cameron N. Dobrowolski, Steven F. Martin, Y. Ranae Robbins, Thomas C. Nay, Lyle M. Boyd, Nathan Coyne, Thomas Wall, Mikeal D. Wittwer, Carl T. Teng, David H.-F. Nucleic Acids Res Molecular Biology Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise ∼16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42–86 bp) genotyping on the LightScanner®. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067°C before calibration to 0.022°C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086°C to 0.032°C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical. Oxford University Press 2008-06 2008-04-29 /pmc/articles/PMC2425497/ /pubmed/18448472 http://dx.doi.org/10.1093/nar/gkn204 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Molecular Biology
Gundry, Cameron N.
Dobrowolski, Steven F.
Martin, Y. Ranae
Robbins, Thomas C.
Nay, Lyle M.
Boyd, Nathan
Coyne, Thomas
Wall, Mikeal D.
Wittwer, Carl T.
Teng, David H.-F.
Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title_full Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title_fullStr Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title_full_unstemmed Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title_short Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
title_sort base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2425497/
https://www.ncbi.nlm.nih.gov/pubmed/18448472
http://dx.doi.org/10.1093/nar/gkn204
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