Evolutionarily distinct polynucleotides for calibration and standardization of quantitative nucleic acid assays

Use of calibrator sequences which are evolutionary unrelated to humans or viruses and microorganisms which infect humans avoids cross-reactivity of primers and probes

Medical & Biotechnology

Template for in vitro transcription reactions to generate the calibrator polynucleotide RNA.

Quantitative real-time polymerase chain reaction (Q-PCR) is used to accurately measure the level of messenger RNA (mRNA) for a polynucleotide of interest in a biological sample. The quantitation of mRNA by Q-PCR is determined in relation to an internal reference gene that is expressed at constant levels in a series of samples. Several internal reference genes, such as beta-actin, GAPDH, and the like, have been used in Q-PCR and are referred to as housekeeping genes. The expression level of these genes has been thought to remain relatively constant across a sample set.

Use of housekeeping genes, however, is not always appropriate since various experimental conditions have been shown to alter the levels of housekeeping genes. In these situations, a known amount of a calibrator polynucleotide that is added to the sample before processing and analysis may be used. The calibrator polynucleotide then becomes the internal reference standard for the Q-PCR assay. The levels of the calibrator polynucleotide are presumed to be independent of the experimental conditions, thereby resulting in an accurate internal standard. But, externally added calibrator polynucleotides are not widely accepted and commercially available as validated universal calibrated polynucleotides. This is at least partially due to the potential for cross-reactivity of primers and probes of calibrator polynucleotides with nucleic acid sequences from humans or organisms that infect humans. These calibration sequences are achieved by a single method— high-stringency assays which reduce but do not eliminate, the probability of interaction between the primers and probes in use and similar sequences in the sample under study.

Army scientists have developed a method to prevent cross-reactivity by an entirely different mechanism – the use of evolutionarily distinct polynucleotide sequences. This approach further obviates the need for a  random sequence generator often used to generate calibrator polynucleotide sequences which can share an unacceptable degree of similarity to sequences from humans. This new method utilizes polynucleotide sequences from plant viroids, bacterial type II introns, and chloroplast type II introns. These sequences from autonomous genetic entities possess sufficient uniqueness to sequences from humans and organisms that infect humans such that they are suitable for use as calibrator polynucleotides and controls in a wide variety of assays.

Applications include:

  • Use as standards or normalization controls in qualitative and quantitative nucleic acid assays including nucleic acid hybridization assays, Q-PCR assays, cDNA and oligonucleotide microarray assays, Northern blotting, and RNase protection assays
  • Use as universal negative controls in an unlimited number of nucleic acid-based assays including DNA footprinting, electrophoretic mobility shift assays (EMSA), and Rapid Amplification of cDNA Ends (RACE)
  • Use in assays to detect and measure markers for disorders and diseases in human and animals

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