Reverse transcription primers and methods of design
View Patent ↗The present invention provides novel algorithms for designing oligonucleotides that do not substantially hybridize to a small group of unwanted transcripts, while hybridizing to most other transcripts. Such oligonucleotides are particularly useful as primers for reverse transcription. The invention also provides compositions containing oligonucleotides that do not substantially hybridize to a small group of unwanted transcripts, while hybridizing to most other transcripts.
1. A method of producing a pool of oligonucleotides that do not hybridize to one or more selected sequences comprising:
selecting at least one exclusion sequence to which hybridization of a pool of oligonucleotides of length n is not desired, wherein the exclusion sequence is an rRNA sequence;
identifying a set of all possible sequences of length n;
identifying a set of all sequences of length n contained in the exclusion sequence;
excluding from the pool of oligonucleotides those oligonucleotides of length n that are identical to a sequence of length n contained in the exclusion sequence;
excluding from the pool of oligonucleotides those oligonucleotides in which the only mismatch to the exclusion sequence is a GU at the 5′ end of the oligonucleotide, and the number of matches to the exclusion sequence wherein the only mismatch is said GU mismatch is greater than 3, or the number of perfect matches to mRNAs is less than 3000; and
wherein a pool of oligonucleotides of length n that do not hybridize to the exclusion sequence are identified and wherein length n is 5-8 nucleotides thereby producing a pool of oligonucleotides that do not hybridize to one or more selected sequences.
2. The method of claim 1 further comprising assessing binding free energy (ΔG) of the oligonucleotides and excluding from the pool of oligonucleotides those oligonucleotides having ΔG below -8kcal/mol.
3. The method of claim 1 , wherein the pool of oligonucleotides is a pool of primers for cDNA synthesis.
4. The method of claim 1 , wherein the method further comprises excluding from the pool of oligonucleotides all those oligonucleotides in which the only mismatch to the exclusion sequence is a GU at the 5′ end of the oligonucleotide.