Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes

David Gresham, Bo Curry, Alexandra Ward, D. Benjamin Gordon, Leonardo Brizuela, Leonid Kruglyak, David Botstein

Research output: Contribution to journalArticle

Abstract

The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate using such a microarray that we detect <90% homozygous SNPs and <80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.

Original languageEnglish (US)
Pages (from-to)1482-1487
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume107
Issue number4
DOIs
StatePublished - Jan 26 2010

Fingerprint

Oligonucleotide Array Sequence Analysis
Freezing
Single Nucleotide Polymorphism
Genome
Research Design
Temperature
Nucleotides
Technology
Gene Expression
DNA Probes
Heterozygote
Saccharomyces cerevisiae
Alleles
Mutation

Keywords

  • DNA/DNA hybridization
  • Melting temperature
  • Probe design
  • Sequence discrimination
  • Single-nucleotide polymorphisms

ASJC Scopus subject areas

  • General

Cite this

Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes. / Gresham, David; Curry, Bo; Ward, Alexandra; Gordon, D. Benjamin; Brizuela, Leonardo; Kruglyak, Leonid; Botstein, David.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 4, 26.01.2010, p. 1482-1487.

Research output: Contribution to journalArticle

Gresham, David ; Curry, Bo ; Ward, Alexandra ; Gordon, D. Benjamin ; Brizuela, Leonardo ; Kruglyak, Leonid ; Botstein, David. / Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes. In: Proceedings of the National Academy of Sciences of the United States of America. 2010 ; Vol. 107, No. 4. pp. 1482-1487.
@article{7ad8ad161a5f438ab7600623981685b6,
title = "Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes",
abstract = "The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate using such a microarray that we detect <90{\%} homozygous SNPs and <80{\%} heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.",
keywords = "DNA/DNA hybridization, Melting temperature, Probe design, Sequence discrimination, Single-nucleotide polymorphisms",
author = "David Gresham and Bo Curry and Alexandra Ward and Gordon, {D. Benjamin} and Leonardo Brizuela and Leonid Kruglyak and David Botstein",
year = "2010",
month = "1",
day = "26",
doi = "10.1073/pnas.0913883107",
language = "English (US)",
volume = "107",
pages = "1482--1487",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "4",

}

TY - JOUR

T1 - Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes

AU - Gresham, David

AU - Curry, Bo

AU - Ward, Alexandra

AU - Gordon, D. Benjamin

AU - Brizuela, Leonardo

AU - Kruglyak, Leonid

AU - Botstein, David

PY - 2010/1/26

Y1 - 2010/1/26

N2 - The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate using such a microarray that we detect <90% homozygous SNPs and <80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.

AB - The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e.g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (Tm) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal Tm by varying the length of probes. We demonstrate using such a microarray that we detect <90% homozygous SNPs and <80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.

KW - DNA/DNA hybridization

KW - Melting temperature

KW - Probe design

KW - Sequence discrimination

KW - Single-nucleotide polymorphisms

UR - http://www.scopus.com/inward/record.url?scp=76549129029&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=76549129029&partnerID=8YFLogxK

U2 - 10.1073/pnas.0913883107

DO - 10.1073/pnas.0913883107

M3 - Article

VL - 107

SP - 1482

EP - 1487

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 4

ER -