Predicting sequence-dependent melting stability of short duplex DNA oligomers

Owczarzy, Richard; Vallone, Peter M.; Gallo, Frank J.; Paner, Teodoro M.; Lane, Michael J.; Benight, Albert S.

doi:10.1002/(sici)1097-0282(1997)44:3<217::aid-bip3>3.0.co;2-y

Cited by 182 publications

(65 citation statements)

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“…Reciprocal melting temperatures were plotted as a function of ln C t . Thermodynamic values were determined from slopes and intercepts of linear fits [25,[33][34][35][36] according to the relationship,…”

Section: Determination Of Thermodynamic Parametersmentioning

confidence: 99%

“…If ΔH°values or ΔS°values differed by more than 15% between both methods, the two-state assumption was considered invalid and the thermodynamic data were not used to determine thermodynamic parameters [25,33,35].…”

Section: Determination Of Thermodynamic Parametersmentioning

confidence: 99%

“…The nearest-neighbor thermodynamic model has been very successful in predicting energetics and extent of hybridization for native DNA [25,26] and RNA duplexes [27,28]. Additional chemical groups can significantly affect duplex stability and melting temperature.…”

Section: Introductionmentioning

confidence: 99%

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Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

Moreira

You

Owczarzy

2015

Biophysical Chemistry

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Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.

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Section: Determination Of Thermodynamic Parametersmentioning

confidence: 99%

Section: Determination Of Thermodynamic Parametersmentioning

confidence: 99%

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Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

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You

Owczarzy

2015

Biophysical Chemistry

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“…Design parameter variables consist of the desired length and predicted t m of an extension primer. Unless stated otherwise all t m and thermodynamic (∆G) values were calculated using nearest-neighbor thermodynamic parameters [24,25]. Primer sequences upstream and downstream adjacent to the SNP site were selected that have the appropriate length and t m characteristics.…”

Section: Extension Primer Designmentioning

confidence: 99%

A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

Vallone

Just

Coble

et al. 2004

International Journal of Legal Medicine

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122

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The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3' end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.

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“…However, the values for ∆H and ∆S available in the literature used in the T m calculation may vary slightly resulting in different predicted T m values [35,36]. For the purpose of this multiplex primer set, the relative comparison of the T m values calculated by Primer3 was sufficient.…”

Section: Check and Compare T Mmentioning

confidence: 99%

Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci

et al. 2003

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The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures ( T(m)) and primer-dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a well-balanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

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Predicting sequence-dependent melting stability of short duplex DNA oligomers

Cited by 182 publications

References 58 publications

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci

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