Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system.
BACKGROUND
With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target.


METHODS
We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses.


RESULTS
RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls).


CONCLUSIONS
The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.
Moser
Michael J
MJ
Eragen Biosciences, Inc., 918 Deming Way, Madison, WI 53717-1944, USA.
Marshall
David J
DJ
Grenier
Jennifer K
JK
Kieffer
Collin D
CD
Killeen
Anthony A
AA
Ptacin
Jerod L
JL
Richmond
Craig S
CS
Roesch
Eric B
EB
Scherrer
Christopher W
CW
Sherrill
Christopher B
CB
Van Hout
Cris V
CV
Zanton
Sara J
SJ
Prudent
James R
JR
D000199:Actins / Q000235:genetics; D001482:Base Composition; D004735:Energy Transfer; D005165:Factor V / Q000235:genetics; D005838:Genotype; D006801:Humans; D009154:Mutation; D009696:Nucleic Acids / Q000235:genetics*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D011516:Prothrombin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D000199:Actins; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; C095381:factor V Leiden; D005165:Factor V; D011516:Prothrombin