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 composition comprising a pool of oligonucleotides that do not hybridize to one or more selected sequences prepared by a process comprising:
selecting at least one exclusion sequence to which hybridization of a pool of oligonucleotides of length n is not desired;
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 is a GU at the 5′ end of the sequence of length n; and
wherein a pool of oligonucleotides of length n that do not hybridize to the exclusion sequence are identified.
2. The composition of claim 1 , wherein the pool of oligonucleotides comprises between 200 to 2,000 non-identical oligonucleotides.
3. The composition of claim 1 , wherein members of the pool of oligonucleotides do not hybridize to a rRNA sequence.
4. A composition comprising a pool of oligonucleotide primers comprising at least 50 non-identical template hybridization sequences of 5 to 8 nucleotides in length and comprising Sequence #s 1 to 379, wherein members of the pool of oligonucleotide primers do not hybridize to a rRNA sequence.
5. The composition of claim 4 , wherein the pool of oligonucleotide primers comprises at least 300 non-identical template hybridization sequences.
6. The composition of claim 4 , wherein the oligonucleotide primers further comprise a promoter recognition sequence.
7. The composition of claim 6 , wherein the oligonucleotide primers further comprise a spacer sequence between the promoter recognition sequence and the template hybridization sequence.
8. The composition of claim 6 , wherein the promoter recognition sequence is a T7 promoter recognition sequence.
9. The composition of claim 4 further comprising one or more of an oligo-dT primer; a reverse transcriptase; or a dNTP mix.
10. The composition of claim 4 further comprising an oligo-dT primer.
11. The composition of claim 10 , wherein the ratio of the oligo-dT primer to the pool of oligonucleotide primers having non-identical template hybridization sequences between about 1:1000 to about 3:2.
12. A composition comprising a pool of oligonucleotide primers comprising at least 50 non-identical template hybridization sequences of 5 to 8 nucleotides in length and comprising Sequence #s 380 to 1152, wherein members of the pool of oligonucleotide primers do not substantially hybridize to a rRNA sequence.
13. The composition of claim 12 , wherein the pool of oligonucleotide primers comprises at least 300 non-identical template hybridization sequences.
14. The composition of claim 12 , wherein the oligonucleotide primers further comprise a promoter recognition sequence.
15. The composition of claim 14 , wherein the oligonucleotide primers further comprise a spacer sequence between the promoter recognition sequence and the template hybridization sequence.
16. The composition of claim 14 , wherein the promoter recognition sequence is a T7 promoter recognition sequence.
17. The composition of claim 12 further comprising one or more of an oligo-dT primer; a reverse transcriptase; or a dNTP mix.
18. The composition of claim 12 further comprising an oligo-dT primer.
19. The composition of claim 18 , wherein the ratio of the oligo-dT primer to the pool of oligonucleotide primers having non-identical template hybridization sequences between about 1:1000 to about 3:2.