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

Abstract

A linker or population of linkers comprising 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 comprising said linker or population of linkers and a target first strand polynucleotide bound to said linker. 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. Provided is a linker instead of G tailing, which can be utilized in a method of preparing a cDNA library, and a method of preparing a cDNA library using said linker.

1 . A linker or a population of linkers comprising an oligonucleotide fixed portion and an oligonucleotide variable portion, 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.  
     
     
         2 . The linker of  claim 1 , wherein the variable portion is prepared at random.  
     
     
         3 . The linker according to  claim 1 , wherein n is an integer ranging from 1 to 10.  
     
     
         4 . The linker according to  claim 1 , wherein n is an integer ranging from 4 to 8.  
     
     
         5 . The linker according to  claim 1 , wherein n is 5 or 6.  
     
     
         6 . The linker according to any one of  claims 1  to  5 , wherein the variable portion is (G) m (N) n-m , and m is an integer ranging from 1 to 3.  
     
     
         7 . The linker according to any one of  claims 1  to  6 , 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 . The linker according to any one of  claims 1  to  7 , wherein the oligonucleotide fixed portion is a single strand or double strand oligonucleotide.  
     
     
         9 . The linker according to  claim 8 , wherein the fixed portion is a double strand oligonucleotide and the single strand variable portion (N)n protrudes outside the double strand fixed portion.  
     
     
         10 . The linker according to any one of  claims 1  to  9 , which is a single strand oligonucleotide having the sequence of SEQ ID NO:1 or SEQ ID NO:3.  
     
     
         11 . The linker according to any one of  claims 1  to  9 , which is a double strand oligonucleotide having the sequence SEQ ID NO:1/SEQ ID NO:2 or SEQ ID NO:3/SEQ ID NO:2.  
     
     
         12 . A double strand oligonucleotide linker or a population of linkers, comprising 
 a) a first oligonucleotide single strand comprising an oligonucleotide single strand fixed portion and an oligonucleotide single strand variable portion, and    b) a second oligonucleotide single strand comprising an oligonucleotide single strand fixed portion annealed to the complementary first oligonucleotide single strand fixed portion (a),    so that the variable portion protrudes outside the double strand fixed portion of the linker;    and wherein the variable portion is represented by the formula (N) n  wherein N is A, C, C, T or U, or their derivatives and n is an integer equal to or higher than 1, and Formula (N)n comprises at least one G.    
     
     
         13 . The linker of  claim 12 , wherein the variable portion is prepared at random.  
     
     
         14 . The linker of  claim 12 , wherein n is an integer ranging from 1 to 10.  
     
     
         15 . The linker of  claim 12 , wherein n is an integer ranging from 4 to 8.  
     
     
         16 . The linker of  claim 12 , wherein n is 5 or 6.  
     
     
         17 . The linker according to any one of  claims 12  to  16 , wherein the variable portion is (G) m (N)n-m , and m is from 1 to 3.  
     
     
         18 . The linker according to any one of  claims 12  to  17 , 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 .  
     
     
         19 . The linker according to any one of  claims 12  to  18 , which has the sequence SEQ ID NO:1/SEQ ID NO:2 or SEQ ID NO:3/SEQ ID NO:2.  
     
     
         20 . A population of linkers comprising at least two linkers according to any one of  claims 1  to  19 .  
     
     
         21 . A population of linkers comprising at least two subpopulations of linkers according to any one of  claims 1  to  20 .  
     
     
         22 . The population of linkers according to any one of  claims 1  to  21 , wherein the fixed portions comprised in all linkers are an oligonucleotide portion having the same sequence.  
     
     
         23 . The population of linkers according to any one of  claims 1  to  22 , 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 which differ each other in the fixed portion sequence.  
     
     
         24 . The population of linkers according to any one of  claims 1  to  23 , wherein the variable portion of the linkers is synthesized at random.  
     
     
         25 . The population of linkers according to any one of  claims 1  to  24 , wherein the sequence of variable portion comprised in each linker differs from each other.  
     
     
         26 . The population of linkers according to any one of  claims 1  to  25 , which is a mixture of the (N)n linker and the (G)m(N)n-m linker, represented as (N)n/(G)m(N)n-m mixture.  
     
     
         27 . The population of linkers according to any one of  claims 1  to  26 , which is a mixture of two or more kinds of linkers having 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  or 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 . The linker or population of linkers according to any one of  claims 1  to  30 , which is prepared by; 
 (a) synthesizing a first oligonucleotide single strand comprising an oligonucleotide single strand fixed portion and an oligonucleotide single strand variable portion,  
 (b) synthesizing a second oligonucleotide single strand comprising an oligonucleotide single strand fixed portion complementary to the first oligonucleotide single strand fixed portion (a), and  
 (c) annealing the first oligonucleotide strand (a) to the second oligonucleotide strand (b) so that the variable portion protrudes outside the double strand fixed linker portion.  
 
     
     
         32 . The linker or population of linkers according to any one of  claims 1  to  31 , wherein the fixed portion has one or more sequences of restriction enzyme sites, recombinational sites, RNA polymerase promoter sites, markers or tags.  
     
     
         33 . The linker or population of linkers according to  claim 32 , wherein the restriction enzyme site is BamHI, XhoI, SstI, SaiI or NotI.  
     
     
         34 . The linker or population of linkers according to  claim 32 , wherein the recombinational site is attB, cre-lox or Gateway.  
     
     
         35 . The linker or population of linkers according to  claim 32 , wherein the polymerase promoter site is selected from the group consisting of T7, T3, K11, SP6 and BA14 RNA polymerase.  
     
     
         36 . The linker or population of linkers according to  claim 32 , wherein the markers are specific sequences or plural oligonucleotides  
     
     
         37 . The linker or population of linkers according to  claim 32 , wherein the tag binds to one or both ends of two strands of the fixed portion, opposite to the variable portion, and said end is a protecting group preventing the formation of binding loop between said linker and an annealed target single strand polynucleotide.  
     
     
         38 . The linker or population of linkers according to  claim 37 , wherein the protecting group is a NH 2  group.  
     
     
         39 . The linker or population of linkers according to  claim 38 , wherein the ends of two strands of the fixed portion of said linker, opposite to the variable portion, are bound together to form a loop so as to prevent said linker from binding to the annealed target single strand polynucleotide.  
     
     
         40 . The population of linkers according to any one of  claims 1  to  39 , which is a population of linkers comprising at least one linker in which the variable oligonucleotide sequence is a specific oligonucleotide sequence, wherein the specific oligonucleotide sequence being able to specifically annealing to the end of at least one defined target polynucleotide to be selected from a population of target polynucleotides.  
     
     
         41 . The linker or population of linkers according to any one of  claims 1  to  40 , which is DNA or RNA or a mixture thereof.  
     
     
         42 . A linker-polynucleotide or population of linker-polynucleotides comprising the linker or population of linkers according to any one of  claims 1  to  41  and the target first strand polynucleotide bound to the linker.  
     
     
         43 . The linker-polynucleotide or population of linker-polynucleotides according to  claim 42 , wherein one end of the target first strand polynucleotide is annealed and/or ligated to the linker.  
     
     
         44 . The linker-polynucleotide or population of linker-polynucleotides according to  claim 42 , wherein one end of the target first strand polynucleotide is annealed to the variable portion of the first strand of the linker and ligated to the second strand fixed portion of the linker.  
     
     
         45 . The linker-polynucleotide or population of linker-polynucleotides according to any one of claims  42 - 44 , wherein the linker is ligated to the target first strand polynucleotide with ligase.  
     
     
         46 . The linker-polynucleotide or population of linker-polynucleotides according to  claim 45 , wherein the ligase is a DNA ligase or RNA ligase.  
     
     
         47 . The linker-polynucleotide or population of linker-polynucleotides according to any one of  claims 42  to  46 , which further comprises a second strand polynucleotide complementary to the target first strand polynucleotide.  
     
     
         48 . The linker-polynucleotide or population of linker-polynucleotides according to  claim 47 , wherein the second strand polynucleotide is synthesized from a free end of the variable portion, with the variable portion acting as a primer.  
     
     
         49 . The single strand or double strand polynucleotide or population of polynucleotides according to any one of  claims 45  to  48 , which is RNA or DNA, or a mixture thereof.  
     
     
         50 . The single strand or double strand polynucleotide or population of polynucleotides according to  claim 49 , wherein DNA is cDNA.  
     
     
         51 . The single strand or double strand polynucleotide or population of polynucleotides according to  claim 50 , which is a long strand, full-coding and/or full-length cDNA.  
     
     
         52 . A vector comprising the linker-polynucleotide according to any one of claims  42 - 51 .  
     
     
         53 . The vector according to  claim 52 , which is a phage, plasmid vector or vector for sequencing.  
     
     
         54 . A method for preparing the linker or population of linkers according to any one of  claims 1  to  41 , which comprises the steps of: 
 (a) synthesizing a first oligonucleotide single strand comprising an oligonucleotide single strand fixed portion and an oligonucleotide single strand variable portion,  
 (b) synthesizing a second oligonucleotide single strand comprising an oligonucleotide single strand fixed portion complementary to the first oligonucleotide single strand fixed portion (a), and  
 (c) annealing the first oligonucleotide strand (a) to the second oligonucleotide strand (b), so that the variable portion protrudes outside the double strand fixed portion of the linker.  
 
     
     
         55 . A method of binding a target single strand polynucleotide to a linker comprising the steps of: 
 i) preparing the linker according to any one of  claims 1  to  41 ; and    ii) annealing the variable portion of said linker to the target single strand polynucleotide.    
     
     
         56 . A method of binding a target single strand polynucleotide to a linker comprising the steps of: 
 i) preparing the linker according to any one of  claims 1  to  41 ; and    ii) annealing the variable portion of the first strand of the linker to the target single strand polynucleotide and ligating the fixed portion of the second strand of the linker to the target single strand polynucleotide.    
     
     
         57 . A method of binding a target single strand polynucleotide or a population of the polynucleotides to a population of linkers comprising the steps of: 
 i) preparing the population of linkers according to any one of  claims 1  to  41 ; and    ii) annealing the variable portion of the population of linkers to the target single strand polynucleotide or to the population of the polynucleotides.    
     
     
         58 . A method of binding a target single strand polynucleotide or a population of the polynucleotides to a population of linkers comprising the steps of: 
 i) preparing the population of linkers according to any one of  claims 1  to  41 ; and    ii) annealing the variable portion of the first strand of said population of linkers to the target single strand polynucleotide or the population of the polynucleotides and ligating the fixed portion of the second strand of the population of linkers to the target single strand polynucleotide or the population of the polynucleotides.    
     
     
         59 . A method of preparing a linker-polynucleotide comprising a linker and a double strand polynucleotide, comprising the steps of: 
 i) annealing the variable portion of the linker according to any one of  claims 1  to  41  to the target first strand polynucleotide, and    ii) synthesizing the second strand polynucleotide complementary to the target single strand polynucleotide.    
     
     
         60 . A method of preparing a linker-polynucleotide comprising a linker and a double strand polynucleotide, comprising the steps of: 
 i) annealing the variable portion of the first strand of the linker according to any one of  claims 1  to  41  to a target single strand polynucleotide and ligating the target single strand polynucleotide to the fixed portion of the second strand of the linker, and    ii) synthesizing the second single strand polynucleotide complementary to said target single strand polynucleotide.    
     
     
         61 . A method of preparing a linker-polynucleotide comprising a linker and a double strand polynucleotide, comprising the steps of: 
 i) annealing the variable portion of the linker or a population of the linkers according to any one of  claims 1  to  41  to a target single strand polynucleotide or a population of the target single strand polynucleotides, and    ii) synthesizing the second strand polynucleotide complementary to said target single strand polynucleotide or a population thereof.    
     
     
         62 . A method of preparing a linker-polynucleotide comprising a linker and a double strand polynucleotide, comprising the steps of: 
 i) annealing the variable portion of the first strand of the linker or a population of the linkers according to any one of  claims 1  to  41  to a target single strand polynucleotide or a population of the target single strand polynucleotides,    ii) ligating the target single strand polynucleotide or the population of the target single strand polynucleotides to the fixed portion of the second strand of the linker or the population of the linkers, and    iii) synthesizing the second single strand polynucleotide(s) complementary to said target single strand polynucleotide(s).    
     
     
         63 . The method according to any one of claims  54 - 62 , wherein one end of the target first strand polynucleotide is annealed to the variable portion of the linker, and ligated to the fixed portion of the second strand of said linker.  
     
     
         64 . The method according to any one of claims  54 - 63 , wherein the variable portion is synthesized at random.  
     
     
         65 . The method according to any one of claims  55 - 64 , wherein the population of linkers comprises at least one linker in which the variable oligonucleotide portion is a specific oligonucleotide portion, this specific oligonucleotide portion being complementary to the end of at least one specific polynucleotide target to be selected from the group of target polynucleotides.  
     
     
         66 . The method according to any one of claims  55 - 65 , wherein ligation is performed by ligase.  
     
     
         67 . The method according to  claim 66 , wherein the ligase is a DNA ligase or RNA ligase.  
     
     
         68 . The method according to  claim 67 , wherein the DNA ligase is T4 DNA ligase or  E. coli  DNA ligase.  
     
     
         69 . The method according to any one of claims  66 - 68 , wherein the ligation is performed in the presence of a ligase-stimulating agent.  
     
     
         70 . The method according to  claim 69 , wherein the ligase-stimulating agent is PEG (polyethylene glycol).  
     
     
         71 . The method according to any one of claims  55 - 70 , wherein the linker and the target first strand polynucleotide and/or the second strand polynucleotide complementary to the target is DNA.  
     
     
         72 . The method according to  claim 71 , wherein the target first strand polynucleotide or the double strand polynucleotide is a long strand, full-coding and/or full-length cDNA.  
     
     
         73 . The method according to  claim 72 , wherein the first strand cDNA is obtained from the Cap trapping at the 5′ end of mRNA.  
     
     
         74 . The method according to  claim 73 , wherein the Cap-trapped cDNA is further normalized or subtracted before or after the ligation to linker.  
     
     
         75 . The method according to any one of claims  55 - 74 , which comprises the step of increasing temperature before annealing the linker to the target first strand polynucleotide and/or after synthesizing polynucleotide second strand.  
     
     
         76 . The method according to  claim 75 , wherein the temperature ranges from 25 to 100° C.  
     
     
         77 . The method according to  claim 76 , wherein the temperature is 65° C.  
     
     
         78 . The method according to any one of claims  55 - 77 , wherein at least one end of the fixed portion of the linker, opposite to the variable portion, is tagged with a protective group.  
     
     
         79 . The method of  claim 78 , wherein the protective group is NH 2 .  
     
     
         80 . The method of any one of claims  55 - 79 , wherein the ends of the two strands of the linker fixed portion are bound together by making a loop.  
     
     
         81 . The method according to any one of claims  55 - 80 , which further comprises the step in which said linker-polynucleotide is cleaved at both ends in restriction enzyme sites and inserted into a vector.  
     
     
         82 . The method according to any one of claims  55 - 80 , which further comprises the step in which said linker-polynucleotide is cleaved remaining blunt ends and inserted into a vector.  
     
     
         83 . A method of marking a polynucleotide library and distinguishing said library, comprising the step of providing a population of linkers, comprising a fixed portion and a variable portion, wherein the fixed portion comprises at least one marker indicating a specific or defined tissue or species, and selecting and separating said library by said specific or defined marker.  
     
     
         84 . The method according to  claim 83 , wherein said linker or population of linkers is the one according to any one of  claims 1  to  41 .  
     
     
         85 . The method according to  claim 83  or  84 , wherein the fixed portions comprised in all linkers among the population of linkers are oligonucleotide portions having the same sequence.  
     
     
         86 . A method of binding a linker or population of linkers to mRNA, which comprises the steps of: 
 (a) treating mRNA with phosphatase and removing phosphate groups from uncapped mRNA,    (b) treating a product of step (a) with pyrophosphatase, which removes the CAP structure from capped mRNA, and    (c) adding an RNA ligase in the presence of the linker according to any one of  claims 1  to  41 .    
     
     
         87 . A method of preparing a linker-polynucleotide, which comprises the steps of: 
 (a) treating mRNA with phosphatase and removing phosphate groups from uncapped mRNA,    (b) treating a product of step (a) with pyrophosphatase, which removes the CAP structure from capped mRNA,    (c) adding an RNA ligase in the presence of the linker according to any one of  claims 1  to  41 , and    (d) adding an oligo dT and synthesizing a polynucleotide complementary to the complete sequence of said mRNA.    
     
     
         88 . The method according to  claim 87 , wherein the polynucleotide complementary to mRNA is cDNA.  
     
     
         89 . The method according to any one of claims  86 - 88 , wherein the phosphatase is bacterial alkaline phosphatase (BAP).  
     
     
         90 . The method according to any one of claims  86 - 88 , wherein the phosphatase is tobacco acid pyrophosphatase (TAP).  
     
     
         91 . The method according to any one of claims  86 - 90 , wherein the linker of step (c) is DNA and further comprising the steps of 
 (e) adding RNase H,    (f) adding DNA polymerase I, and    (g) synthesizing a cDNA strand.    
     
     
         92 . A method of binding a linker or a population of linkers according to any one of  claims 1  to  41  to a target single strand polynucleotide or population of polynucleotides comprising the step of adding RNA ligase.  
     
     
         93 . The method according to  claim 92 , which further comprises the addition of an oligonucleotide primer or population of primers complementary to the variable portion of the linker or population of linkers.  
     
     
         94 . The method according to  claim 93 , wherein the primer comprised in the population of primers is randomly synthesized.  
     
     
         95 . A method of preparing a second strand polynucleotide, which comprises the step according to any one of claims  92 - 94  and further synthesizing the second strand polynucleotide complementary to a target polynucleotide.  
     
     
         96 . A method of preparing a DNA/RNA hybrid, which comprises the steps of: 
 i) providing a full-length/coding or long poly-A mRNAs,    ii) ligating and annealing said mRNA to the linker according to any one of  claims 1  to  41 , the linker comprising a first restriction enzyme site,    iii) annealing an oligo dT-primer comprising a second restriction enzyme site to the mRNA,    iv) synthesizing a cDNA strand,    v) isolating the hybrid by using restriction enzymes which recognize the two specific restriction enzyme sites introduced, and    vi) cloning.    
     
     
         97 . A method of preparing a linker-polynucleotide product comprising a linker and a single strand polynucleotide, comprising annealing the variable portion of the linker according to the present invention to the target first strand polynucleotide.  
     
     
         98 . (New) A linker or a population of linkers comprising an oligonucleotide fixed portion and an oligonucleotide variable portion, 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, wherein the fixed portion has one or more sequences of recombinational sites, RNA polymerase promoter sites, markers or tags.  
     
     
         99 . (New) The linker or population of linkers according to  claim 98 , wherein the recombinational site is attB, cre-10× or Gateway.  
     
     
         100 . (New) The linker or population of linkers according to  claim 98 , wherein the polymerase promoter site is selected from the group consisting of T7, T3, K11, SP6 and BA14 RNA polymerase.  
     
     
         101 . (New) The linker or population of linkers according to  claim 98 , wherein the markers are specific sequences or plural oligonucleotides  
     
     
         102 . (New) The linker or population of linkers according to  claim 98 , wherein the tag binds to one or both ends of two strands of the fixed portion, opposite to the variable portion, and said end is a protecting group preventing the formation of binding loop between said linker and an annealed target single strand polynucleotide.  
     
     
         103 . (New) The linker or population of linkers according to  claim 102 , wherein the protecting group is a NH 2  group.  
     
     
         104 . (New) The linker or population of linkers according to  claim 103 , wherein the ends of two strands of the fixed portion of said linker, opposite to the variable portion, are bound together to form a loop so as to prevent said linker from binding to the annealed target single strand polynucleotide.