To the top

Page Manager: Webmaster
Last update: 9/11/2012 3:13 PM

Tell a friend about this page
Print version

Properties of targeted pr… - University of Gothenburg, Sweden Till startsida
Sitemap
To content Read more about how we use cookies on gu.se

Properties of targeted preamplification in DNA and cDNA quantification

Review article
Authors Daniel Andersson
Nina Akrap
David Svec
T. E. Godfrey
M. Kubista
Göran Landberg
Anders Ståhlberg
Published in Expert Review of Molecular Diagnostics
Volume 15
Issue 8
Pages 1085-1100
ISSN 1473-7159
Publication year 2015
Published at Institute of Biomedicine, Department of Pathology
Sahlgrenska Cancer Center
Institute of Biomedicine
Pages 1085-1100
Language en
Links dx.doi.org/10.1586/14737159.2015.10...
Keywords experimental design, multiplex PCR, preamplification, primer-pools, quantitative real-time PCR, REAL-TIME PCR, POLYMERASE-CHAIN-REACTION, BOVINE SERUM-ALBUMIN, EMBRYONIC STEM-CELLS, SINGLE-CELL, GENE-EXPRESSION, QUANTITATIVE PCR, RT-PCR, AMPLIFICATION, BETAINE, Pathology, EVET E, 1995, NUCLEIC ACIDS RESEARCH, V23, P3343, ENG S, 1994, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF
Subject categories Basic Medicine

Abstract

Objective: Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). Methods: To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. Result: The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. Conclusion: On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

Page Manager: Webmaster|Last update: 9/11/2012
Share:

The University of Gothenburg uses cookies to provide you with the best possible user experience. By continuing on this website, you approve of our use of cookies.  What are cookies?