Oligonucleotide linkers comprising a variable cohesive portion and method for the preparation of polynucleotide libraries by using said linkers

Abstract

The present invention relates to a linker or population of linkers that include an oligonucleotide fixed portion and an oligonucleotide variable portion represented by formula (N)n, wherein N is A, C, G, T or U, or their derivatives, and n is an integer equal to or higher than 1. A linker-polynucleotide or a population of linker-polynucleotides of the invention may be constituted by said linker or population of linkers and a target first strand polynucleotide bound to said linker. The invention also encompasses a method of preparing said linker or population of linkers and a method of preparing a linker-polynucleotide using said linker or population of linkers. The linkers or polynucleotide-linkers of the invention can be used in a method of preparing a cDNA library.

1 . A population of linkers comprising an oligonucleotide fixed portion and an oligonucleotide variable portion, the fixed portion being a double stranded oligonucleotide and the variable portion being represented as Formula (N)n wherein N is A, C, G, T or U, or their derivatives, and n is an integer equal to or higher than 1, and if n is an integer equal to or higher than 2, the nucleotides (N) may be the same or different from each other, and Formula (N)n comprises at least one G, said population of linkers being comprised of linkers of the same sequence or being comprised of linkers of at least two different sequences. 
     
     
         2 . The population of linkers of  claim 1 , wherein the variable portion has a randomized sequence. 
     
     
         3 . The population of linkers according to  claim 1 , wherein n is an integer ranging from 1 to 10. 
     
     
         4 . The population of linkers according to  claim 1 , wherein n is an integer ranging from 4 to 8. 
     
     
         5 . The population of linkers according to  claim 1 , wherein n is 5 or 6. 
     
     
         6 . The population of linkers according to  claim 1 , wherein the variable portion is (G) m (N) n-m , and m is an integer ranging from 1 to 3. 
     
     
         7 . The population of linkers according to  claim 1 , wherein the variable portion is GN 4 , GN 5 , G 2 N 3 , G 2 N 4 , G 3 N 2 , G 3 N 3 , N 5  or N 6 . 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The population of linkers according to  claim 1 , which comprises an oligonucleotide having the sequence of SEQ ID NO:1 or SEQ ID NO:3. 
     
     
         11 - 21 . (canceled) 
     
     
         22 . The population of linkers according to  claim 1 , wherein the fixed portions comprised in all linkers are an oligonucleotide portion having the same sequence. 
     
     
         23 . The population of linkers according to  claim 1 , comprising two or more subpopulations of linkers, wherein one subpopulation of linkers comprise linkers in which the fixed portion is an oligonucleotide portion having the same sequence, and other subpopulations of linkers differ from each other in the fixed portion sequence. 
     
     
         24 . The population of linkers according to  claim 1 , wherein the variable portion of the linkers has a randomized sequence. 
     
     
         25 . The population of linkers according to  claim 1 , wherein the sequence of the variable portion comprised in each linker differs from each other. 
     
     
         26 . The population of linkers according to  claim 1 , that comprises linkers having at least two different sequences, at least one linker having from 1 to 3 G residues at the 5′ end. 
     
     
         27 . The population of linkers according to  claim 1 , which is a mixture of two or more linkers having different sequences, said sequences comprising any variable portion selected from the group consisting of GN 4 , GN 5 , G 2 N 3 , G 2 N 4 , G 3 N 2 , G 3 N 3 , N 5  and N 6 . 
     
     
         28 . The population of linkers according to  claim 27 , which is a N 6 /GN 5  mixture, N 6 /G 2 N 4  mixture or N 6 /G 3 N 3  mixture. 
     
     
         29 . The population of linkers according to  claim 28 , wherein the mixing ratio ranges from 0:1 to 1:0. 
     
     
         30 . The population of linkers according to  claim 29 , wherein the mixing ratio is 1:4. 
     
     
         31 .- 104 . (canceled)