Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets.
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.
Benavides
G R
GR
Department of Zoology, University of Georgia, Athens 30602, USA.
Hubby
B
B
Grosse
W M
WM
McGraw
R A
RA
Tarleton
R L
RL
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000592:standards*; D012333:RNA, Messenger / Q000235:genetics; D014349:Trypanosoma cruzi / Q000235:genetics
D018076:DNA, Complementary; D016054:DNA, Protozoan; D015345:Oligonucleotide Probes; D012333:RNA, Messenger