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J Immunol Methods. 1995 Apr 26;181(2):145-56.

Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets.

Author information

1
Department of Zoology, University of Georgia, Athens 30602, USA.

Abstract

We describe a simple and inexpensive method for the construction of multi-competitor molecules for use as internal standards in quantitative RT-PCR. The construction involves the linking and annealing of 20mer PCR primers with complementary 40mers using either a step-wise or bulk process. The entire construct is then ligated and amplified by PCR prior to cloning. Using this approach, we have constructed a gene containing priming sites for 18 different products of immunological interest, including murine cytokines and cell surface markers, as well as murine beta-actin and T. cruzi rRNA. The cost of production of the competitor is minimized by use of a high-throughput multi-oligonucleotide synthesizer for production of the individual components of the synthetic gene, and by use of the same oligonucleotides in gene construction and as primers for the RT-PCR reactions. This procedure can be applied to the production of other polycompetitor molecules as well as to the construction of other types of synthetic genes.

PMID:
7745244
DOI:
10.1016/0022-1759(94)00339-x
[Indexed for MEDLINE]

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