Now we add in init = 4.1 cal/mole + 4.1cal/mole+ - 310.821cal/mole Here is an example of a Tm calculation using: Salt equiv: 538.18 mM If the primer begins with a G orĪ C, then the init G-C correction is added, otherwise the init A-T correction is added. The dH is found by adding up the (LNA(TM)/DNA) di-nucleotide pairs values from the table belowĪnd DNA/DNA dinulceotide pair values from Santa Lucia values. Melting Temperature Calculation For LNA(TM) ProbesĬ - Primer concentration: 50 nM (acceptable range 0.1 - 100 nM) Mg++:1.8 mM for TaqMan(R) design (acceptable range 0 - 100 mM) Tm for primer as predicted by Primer Premier using Breslaur values: 52.5 oCĭinucleotide Sequence H kcal/mol S cal/k mol NaEqiv = Na+ + 4 x sqrt(Mg++) (all concentrations molar) The Tm for a primer is calculated from its H, S, R the gas constant (1.987 cal/kmol), and C the Number of nucleotide pairs in the primer (primer length - 1):ĭS (salt corrected) = dS (1 M NaCl) + 0.368 * N * ln () An addition salt correction term is added where N is the The same is done for the base at the end of primer. If the primerīegins with a G or a C, then the init G-C correction is added, otherwise the init A-T correction isĪdded. The dH is found by adding up all the di-nucleotide pairs values from the table below. = Monovalent ion concentration + 4 X (Free Mg2+)1/2 (all in molar concentration) Monovalent ion concentration: 50 mM (acceptable range 0 - 1000 mM) Equivalent Algorithm: The sodium equivalent is calculated by: Mg++: 3.0 mM for beacon and 5.0mM for TaqMan(R) design (acceptable range 0 - 100 mM) The Tm for the primer is calculated using the following:Ĭ - Primer concentration: 0.25 nM (acceptable range 0.1 - 100 nM) Untergasser A et al (2012) Primer3-new capabilities and interfaces.Formula for Melting Temperature Calculation Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Adv Biomed Res 3:85Īndersen C et al (2006) Equal performance of TaqMan, MGB, Molecular Beacon and SYBR Green-based detection assays in detection and quantification of roundup ready soybean. Tajadini M et al (2014) Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Maeda H et al (2003) Quantitative real-time PCR using TaqMan and SYBR Green Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. Paudel D et al (2001) Comparison of real-time SYBR Green dengue assay with real-time TaqMan RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection. Int Drug Disc:18–24īustin S, Nolan T (2004) Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. Nucleic Acids Res 39(9):e63ĭ’haene B, Hellemans J (2010) The importance of quality control during qPCR data analysis. Vermeulen J et al (2001) Measurable impact of RNA quality on gene expression results from quantitative PCR. Karlen Y et al (2007) Statistical significance of quantitative PCR. Quantitative real-time polymerase chain reaction.Here we have shown how to use some freely available web-based software programs (such as Primerquest ®, Unafold ®, and Beacon designer ®) to design qPCR primers. Freely available software could be used for ideal qPCR primer design. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. However, success of qPCR depends on the optimal primers used. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon ®, SYBR Green ®, and Taqman ®. In qPCR, a reporter dye system is used which intercalates with DNA’s region of interest and detects DNA amplification. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized “real-time” using a computer. Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression.
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