Abstract
To simplify pyrosequencing protocol, we improved linear-after-the-exponential-PCR (imLATE-PCR) for yielding single-stranded DNA templates directly for pyrosequencing reaction. However, it is very critical for designing qualified primers for imLATE-PCR. To achieve a high amplification efficiency in imLATE-PCR, the polymerase preference index (PPI) value together with conventional melting temperature-dependent method were employed to design the primers. Issues frequently occurring in pyrosequencing based on conventional LATE-PCR, such as a low signal intensity or a high non-specific peak, were well solved by the PPI technology. The new strategy is validated by using pyrosequencing for various application, including resequencing, quantitative SNP genotyping, and relative gene expression analysis. Successful detection of gene sequence of genetically modified organisms, copy number variations of samples from Down’s syndrome (trisomy-21) patients, and gene expression levels in tissues from patients of breast cancer demonstrates the feasibility of this method for real application. Compared with pyrosequencing based on conventional LATE-PCR, the merits lie on a flexible primer design, increased yield of ssDNA, and good quality of pyrograms.
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Song, Q., Qi, X., Shen, Y., Jin, Y., Zou, B., Zhou, G. (2016). Using Polymerase Preference Index to Design imLATE-PCR Primers for an Efficient Pyrosequencing. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_13
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DOI: https://doi.org/10.1007/978-1-4939-3308-2_13
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3306-8
Online ISBN: 978-1-4939-3308-2
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