By Guohua Zhou, Qinxin Song
The goal of this ebook is to enhance pyrosequencing protocols in addition to instrumentation for higher medical use by way of describing advancements and novel functions of pyrosequencing technology. Divided into 5 elements, the book’s thirty chapters discover advances in pyrosequencing template training, pyrosequencing know-how suggestions, multiplex pyrosequencing in keeping with barcodes, the miniaturization of pyrosequencing apparatus, in addition to numerous functions. As a part of the Springer Protocols application, chapters include the type of element and useful implementation suggestion to assure winning ends up in the lab.
Comprehensive and thorough, Advances and scientific perform in Pyrosequencing serves as a helpful reference for researchers who're engaged in customized drugs, disorder keep an eye on, and DNA analysis in different different fields.
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Additional resources for Advances and Clinical Practice in Pyrosequencing (Springer Protocols Handbooks)
For the advantages of accuracy, flexibility, and parallel processing [2–5], pyrosequencing has been widely used for DNA resequencing [6–8], genotyping [9, 10], DNA methylation , and gene expression analysis . In conventional pyrosequencing protocol, a biotin-modified PCR primer and streptavidin-coated beads are required to prepare ssDNA template prior to pyrosequencing. 1007/978-1-4939-3308-2_4, © Springer Science+Business Media New York 2016 31 32 Qinxin Song et al. prepare ssDNA are expensive  and multiple steps that are needed to prepare the template increase the risk of DNA contamination in the air.
2. PCR mixture denatured at 94 °C for 15 min and followed by 35 cycles (94 °C for 40 s; 55 °C for 40 s; 72 °C for 1 min). After the cycle reaction, the product was incubated at 72 °C for 10 min and held at 4 °C before use. 2 Treatment of dsDNA with Nicking Endonucleases 1. PCR products were purified by the commercially available PCR purification kit or enzymatically treated by apyrase coupled with ATP sulfurylase (see Note 1). 2. BstNBI in a 1× reaction buffer (see Note 2). 5–1 h with 5–10 U of nicking endonuclease (see Note 3).
Most importantly, the length of amplicons should be short enough to ensure that Tm difference between the amplicon and the excess primer is less than 13 °C. Usually, an SNP loci of interest is fixed, so a difficulty may occur in designing qualified LATE-PCR primers at GC-rich or AT-rich regions. The main problem of low amplification efficiency of LATE-PCR is due to the competition of excess primer with the amplicon strand. As amplicon strand is increasing in the concentration along with the progress of PCR, while the excess primer is decreasing in the concentration, the yield of ssDNA should not be high if the amplicon is longer [3–6].