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Properties of targeted preamplification in DNA and cDNA quantification

Forskningsöversiktsartikel
Författare Daniel Andersson
Nina Akrap
David Svec
T. E. Godfrey
M. Kubista
Göran Landberg
Anders Ståhlberg
Publicerad i Expert Review of Molecular Diagnostics
Volym 15
Nummer/häfte 8
Sidor 1085-1100
ISSN 1473-7159
Publiceringsår 2015
Publicerad vid Institutionen för biomedicin, avdelningen för patologi
Sahlgrenska Cancer Center
Institutionen för biomedicin
Sidor 1085-1100
Språk en
Länkar dx.doi.org/10.1586/14737159.2015.10...
Ämnesord 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
Ämneskategorier Medicinska grundvetenskaper

Sammanfattning

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.

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