+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Determination of melting temperature for variant detection using dHPLC: a comparison between an empirical approach and DNA melting prediction software



Determination of melting temperature for variant detection using dHPLC: a comparison between an empirical approach and DNA melting prediction software



Genetic Testing 6(3): 169-176



Detection of DNA sequence variants by the use of denaturing high-performance liquid chromatography (dHPLC) is a relatively new method (Underhill et al., 1997) and has distinct advantages over other methods such as single-strand conformation polymorphism (SSCP), direct sequencing, and DNA chip hybridization. The dHPLC-based single-nucleotide polymorphism (SNP) screening relies on different DNA thermodynamic properties between perfectly matched base pairs in homoduplex molecules and single base-pair mismatches in heteroduplex DNAs. Separation of the two forms of duplex DNAs by dHPLC is based on ionic forces between the negatively charged DNA and the hydrophobic stationary phase, which consists of C(18) chains on PSDVB (polystyrene-divinylbenzene) beads coated with a positively charged ion-pairing agent (TEAA, triethylammonium acetate). Removal of the DNA from the TEAA-coated beads is dependent upon a mobile organic phase, in the form of a linear acetonitrile gradient. The major factor that influences the success of dHPLC to detect sequence variation is the thermal stability of the duplex DNA, which is determined by the melting temperature (TM(50)), where 50% of the DNA strand is single stranded and 50% is double stranded. The TM(50) predicts the best probability of detecting a single base-pair change based on the altered thermodynamics it imparts to the DNA duplex. Generally, there are two ways to determine this melting temperature, either empirically or with the aid of predictive DNA melting analysis software. Such programs include the DNAMelt program located on the Stanford University DNA Sequencing and Technology Center website, MeltCalc (Schutz and von Ahsen 1999), and WAVEMAKER, the proprietary melting analysis software provided with the Transgenomic WAVE dHPLC system. The goal of the current study was to determine whether currently available predictive DNA melting programs could be used to increase efficiency and throughput of SNP detection. A wide range of amplicons, differing in both size and GC composition, were selected for analysis to simulate the broad spectrum of PCR products that may be encountered during a large-scale dHPLC screening project.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 010441944

Download citation: RISBibTeXText

PMID: 12490056

DOI: 10.1089/109065702761403324


Related references

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves. Analytical Biochemistry 479: 28-36, 2016

Research on a reference substance for determination of melting temperature after Ph. Helv. VI and Ph. Eur. I. 1. General information on crystals, melting and definition of melting point. Pharmaceutica Acta Helvetiae 48(11): 639-653, 1973

Reference compounds for determination of melting temperature according to Ph. Helv. VI and Ph. eur. I. 4. Determination of purity and melting point with differential scanning calorimetry--results and discussion. Pharmaceutica Acta Helvetiae 49(3-4): 102-107, 1974

Reference substances for determination of melting temperatures according to Ph. Helv. VI and Ph. Eur. I. 3. Determination of melting point and melting range and results. Pharmaceutica Acta Helvetiae 49(2): 57-65, 1974

A self-consistent estimation method of melting condition based on major elements in volcanic rocks; degree of melting, pressure, H2 O content and melting temperature. Eos, Transactions, American Geophysical Union 89.53, Suppl., 2008

Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination. Journal of Microbiological Methods 87(2): 195-201, 2012

An empirical approach to estimate melting temperature and its pressure dependence of some rocks of Oman ophiolite suite. Mineralogical Magazine 75.3: 446, 2011

Quantifying variant differences in DNA melting curves: Effects of length, melting rate, and curve overlay. Analytical Biochemistry 539: 90-95, 2017

Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes. Bmc Bioinformatics 8: 107, 2007

uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics 27(7): 1019-1020, 2011

Validation of a melting fraction-based effective thermal conductivity correlation for prediction of melting phase change inside a sphere. International Journal of Thermal Sciences 142: 247-257, 2019

In silico prediction of the melting points of ionic liquids from thermodynamic considerations: a case study on 67 salts with a melting point range of 337 degrees C. Journal of Physical Chemistry. B 114(34): 11133-11140, 2010

Investigation of melting and resolidification of Sm1.8Ba2.4Cu3.4Ox in Ba-Cu-O melt at the temperature below its melting point. Journal of Materials Science Letters 19(14): 1253-1254, 2000

Discovering rare variants by use of melting temperature shifts seen in melting curve analysis. Clinical Chemistry 51(8): 1331-1332, 2005

The effect of melting temperature and a detergent additive on the melting behavior of propyphenazone-containing suppositories. Die Pharmazie 43(1): 51-52, 1988