Please use this identifier to cite or link to this item: https://doi.org/10.1016/j.mimet.2009.03.001
DC FieldValue
dc.titleQuantitative effects of position and type of single mismatch on single base primer extension
dc.contributor.authorWu, J.-H.
dc.contributor.authorHong, P.-Y.
dc.contributor.authorLiu, W.-T.
dc.date.accessioned2014-10-08T08:33:28Z
dc.date.available2014-10-08T08:33:28Z
dc.date.issued2009-06
dc.identifier.citationWu, J.-H., Hong, P.-Y., Liu, W.-T. (2009-06). Quantitative effects of position and type of single mismatch on single base primer extension. Journal of Microbiological Methods 77 (3) : 267-275. ScholarBank@NUS Repository. https://doi.org/10.1016/j.mimet.2009.03.001
dc.identifier.issn01677012
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/87607
dc.description.abstractSingle mismatch (MM) present at the region where primer binds onto the template strand can greatly affect the PCR efficacy. Earlier studies revealed that PCR or primer extension is hindered by a single MM at the primer 3′ end. The MMs located at other positions within a primer also have similar performance, but to what extent they can decrease the efficiency is not clear. In this study, a modified single base extension assay was used to systematically compare the extension efficiencies between a perfect-matched (PM) primer and its single-MM primers with all possible MM types. The extension efficiencies of single-MM primers, which were generally lower or equivalent to that of the PM primer, were observed to strongly depend on the MM location and/or type. Due to the enzymatic activity, single MMs present at the last 3-4 positions from the primer 3′ end exhibited zero or minimal (< 3.9%) extension efficiencies. For those MMs at positions 5 onward from primer 3′ end where was affected mainly by the primer-target binding stability, an increasing trend in extension efficiency with the highest (i.e., 69.3%) occurring at the primer 5′ end was observed to significantly correlate in an inverse relationship with the duplex stability (i.e., difference of melting temperature) under a empirically polynomial equation, y = - 0.0731x3 + 2.2519x2 - 22.617x + 76.691 (R2 = 0.5318). It was further shown that the extension efficiencies of these MM types could be improved with a factor of 3.25 on average in relation to the decrease in the annealing temperature by 7 °C. On the other hand, substitution of a less selective inosine nucleotide did not convincingly improve the extension efficiency. Overall findings obtained could further improve the rational design of oligonucleotide primers in various microbiological studies that involve the use of PCR techniques. Crown Copyright © 2009.
dc.description.urihttp://libproxy1.nus.edu.sg/login?url=http://dx.doi.org/10.1016/j.mimet.2009.03.001
dc.sourceScopus
dc.subjectHybridization
dc.subjectPrimer extension
dc.subjectSingle nucleotide mismatch
dc.typeArticle
dc.contributor.departmentDIVISION OF ENVIRONMENTAL SCIENCE & ENGG
dc.description.doi10.1016/j.mimet.2009.03.001
dc.description.sourcetitleJournal of Microbiological Methods
dc.description.volume77
dc.description.issue3
dc.description.page267-275
dc.description.codenJMIMD
dc.identifier.isiut000267084000003
Appears in Collections:Staff Publications

Show simple item record
Files in This Item:
There are no files associated with this item.

SCOPUSTM   
Citations

101
checked on May 16, 2022

WEB OF SCIENCETM
Citations

96
checked on May 16, 2022

Page view(s)

127
checked on May 12, 2022

Google ScholarTM

Check

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.