Reverse transcription primers and methods of design

Abstract

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 designing 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;   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 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 −8 kcal/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 exclusion sequence is an rRNA sequence. 
     
     
         5 . The method of  claim 1 , wherein the length n is 5 to 8 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein the length n is 5 nucleotides and the pool of oligonucleotides comprises between 100 to 400 non-identical oligonucleotides. 
     
     
         7 . The method of  claim 1 , wherein the length n is 6 nucleotides and the pool of oligonucleotides comprises between 200 to 2,000 non-identical oligonucleotides. 
     
     
         8 - 12 . (canceled) 
     
     
         13 . A composition comprising a pool of oligonucleotide primers comprising at least 50 non-identical template hybridization sequences of 5 to 8 nucleotides in length, wherein the pool of oligonucleotide primers does not substantially hybridize to a rRNA sequence. 
     
     
         14 . The composition of  claim 13 , wherein the pool of oligonucleotide primers comprises at least 300 non-identical template hybridization sequences. 
     
     
         15 . The composition of  claim 13 , wherein the pool of oligonucleotide primers comprises Sequence #s 1 to 379. 
     
     
         16 . The composition of  claim 13 , wherein the pool of oligonucleotide primers comprises Sequence #s 380 to 1152. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . The composition of  claim 13  further comprising an oligo-dT primer. 
     
     
         22 . The composition of  claim 21 , 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. 
     
     
         23 . A kit, in a suitable container, comprising a plurality of oligonucleotide primers comprising 50 and 2,500 non-identical template hybridization sequences of 4 to 11 nucleotides in length. 
     
     
         24 . The kit of  claim 23 , wherein the oligonucleotide primers comprise Sequence ID NOs. 1 to 1152. 
     
     
         25 . The kit of  claim 23 , wherein the of oligonucleotide primers further comprise a promoter recognition sequence. 
     
     
         26 . The kit of  claim 23 , further comprising one or more of an oligo-d(T) primer; a reverse transcriptase; a buffer; a dNTP mix; an RNA polymerase; a single strand binding protein; ethylenediaminetetraacetic acid (EDTA); a promoter-oligo-d(T) primer; a ribonuclease inhibitor; a DNA polymerase; RNase H; nuclease free water; ATP; CTP; GTP; UTP; TTP; DNase I; an aRNA filter cartridge; a cDNA filter cartridge; or collection tubes.