By Guohua Zhou, Qinxin Song
The objective of this booklet is to enhance pyrosequencing protocols in addition to instrumentation for higher scientific use by way of describing advancements and novel functions of pyrosequencing expertise. Divided into 5 components, the book’s thirty chapters discover advances in pyrosequencing template instruction, pyrosequencing expertise suggestions, multiplex pyrosequencing in line with barcodes, the miniaturization of pyrosequencing apparatus, in addition to numerous functions. As a part of the Springer Protocols application, chapters comprise the type of aspect and functional implementation suggestion to assure profitable ends up in the lab.
Comprehensive and thorough, Advances and medical perform in Pyrosequencing serves as a important reference for researchers who're engaged in customized medication, affliction keep an eye on, and DNA prognosis in several different fields.
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Additional info for Advances and Clinical Practice in Pyrosequencing
As the excess primer is longer in length, its Tm could be very close to Tm of amplicon, resulting in a very higher rate of linear amplification, which is a limiting process for yielding ssDNA. Therefore the length of amplicon could be longer, and the choice for primer sequences could be more. One potential issue of imLATE-PCR is the poor specificity due to a lower annealing temperature in the phase of exponential amplification. To check this issue, and to verify whether or not imLATE-PCR is effective in the amplification of various lengths of amplicons, different primer sets are used to amplify the BRCA1 gene related to human breast cancer by symmetric PCR, LATE-PCR, and imLATE-PCR, respectively.
Chin J Anal Chem 36:775–780 13. Song Q, Wu H, Feng F, Zhou G, Kajiyama T, Kambara H (2010) Pyrosequencing on nicked dsDNA generated by nicking endonucleases. Anal Chem 82:2074–2081 14. Sanchez JA, Pierce KE, Rice JE, Wangh LJ (2004) Linear-after-the-exponential (LATE)PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc Natl Acad Sci U S A 101:1933–1938 15. Salk JJ, Sanchez JA, Pierce KE, Rice JE, Soares KC, Wangh LJ (2006) Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.
Kroneis T, Geigl JB, El-Heliebi A, Auer M, Ulz P, Schwarzbraun T, Dohr G, Sedlmayr P (2011) Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification. Clin Chem 57:1032–1041 5. Sanchez JA, Pierce KE, Rice JE, Wangh LJ (2004) Linear-after-the-exponential (LATE)PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc Natl Acad Sci U S A 101:1933–1938 6. Pierce KE, Sanchez JA, Rice JE, Wangh LJ (2005) Linear-After-The-Exponential (LATE)PCR: primer design criteria for high yields of 7.
Advances and Clinical Practice in Pyrosequencing by Guohua Zhou, Qinxin Song