Abstract
BackgroundComputer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. However, even for regions that are equally conserved, their potential utility as hybridization targets varies. Mismatches in sequence variants are more disruptive in some duplexes than in others. Additionally, the propensity for self-interactions amongst oligonucleotides targeting conserved regions differs and the structure of target regions themselves can also influence hybridization efficiency. There is a need to develop software that will employ thermodynamic selection criteria for finding optimal hybridization targets in related sequences.ResultsA new scheme and new software for optimal detection of oligonucleotide hybridization targets common to families of aligned sequences is suggested and applied to aligned sequence variants of the complete HIV-1 genome. The scheme employs sequential filtering procedures with experimentally determined thermodynamic cut off points: 1) creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotide targets in the analyses; 2) selection of DNA oligonucleotides that have pairing potential, greater than a defined threshold, with all variants of aligned RNA sequences; 3) elimination of DNA oligonucleotides that have self-pairing potentials for intra- and inter-molecular interactions greater than defined thresholds. This scheme has been applied to the HIV-1 genome with experimentally determined thermodynamic cut off points. Theoretically optimal RNA target regions for consensus oligonucleotides were found. They can be further used for improvement of oligo-probe based HIV detection techniques.ConclusionsA selection scheme with thermodynamic thresholds and software is presented in this study. The package can be used for any purpose where there is a need to design optimal consensus oligonucleotides capable of interacting efficiently with hybridization targets common to families of aligned RNA or DNA sequences. Our thermodynamic approach can be helpful in designing consensus oligonucleotides with consistently high affinity to target variants in evolutionary related genes or genomes.
Highlights
Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants
Beside PCR, such detection can be performed using strand displacement amplification (SD) [1,2], transcription-mediated amplification (TMA) [3], nucleic acid sequence-based amplification (NASBA) [4], hybridization protection assay [5], branched DNA signal amplification [6,7], in situ hybridization [8,9] or other techniques that are currently being developed and require oligonucleotides interacting with RNA or DNA as a basic step
The scheme developed for discrimination of conserved regions in multiple sequence variants is based on their potential to serve as efficient hybridization targets for oligonucleotides and involves several steps: First, creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotides in the analyses
Summary
Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. Finding optimal targets for oligonucleotides in multiple variants of related sequences is useful for a number of practical tasks. Beside PCR, such detection can be performed using strand displacement amplification (SD) [1,2], transcription-mediated amplification (TMA) [3], nucleic acid sequence-based amplification (NASBA) [4], hybridization protection assay [5], branched DNA signal amplification [6,7], in situ hybridization [8,9] or other techniques that are currently being developed and require oligonucleotides interacting with RNA or DNA as a basic step. To develop more sensitive and far reaching detection assays, it is important to select HIV-1 RNA target regions where mutations are least disruptive for potential duplex formation with complementary oligonucleotides
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