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DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using DNA polymerase terminating dideoxynucleotides, and the ratio of C to T is determined quantitatively.
NetPrimer is a gratis web-based tool used for analysing primers used in PCR to amplify a DNA sequence. [2] The software predicts the melting temperature of the primers using the nearest neighbor thermodynamic algorithm. The accurate prediction of the melting temperature (Tm) is one of the most important factors that governs the success of a PCR ...
Hexamer primers are sequences composed of six random nucleotides. For MDA applications, these primers are usually thiophosphate-modified at their 3’ end to convey resistance to the 3’–5’ exonuclease activity of Ф29 DNA polymerase. MDA reactions start with the annealing of such primers to the DNA template followed by polymerase-mediated ...
This is due to there being two primers, the initiating primer (IP) and the Vectorette primer (VP), that act in different stages. During the first part, the IP works on amplifying the primer extension while the VP remains hybridized with the product; thus, any background amplification is not carried out at this stage.
Primer extension can be used to determine the start site of transcription (the end site cannot be determined by this method) by which its sequence is known. This technique requires a radiolabelled primer (usually 20 - 50 nucleotides in length) which is complementary to a region near the 3' end of the mRNA.
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The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, [6] as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results ( Electronic PCR ) may be performed to assist in primer design by giving melting and annealing temperatures, etc. [ 7 ]
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