Strand displacement stop (sds) ligation

Abstract

A method of ligating DNA molecules, wherein the DNA molecules are in a hybrid with an RNA molecule, including the steps of providing DNA molecules that are in a RNA:DNA hybrid with an RNA molecule, and ligating the DNA molecules to each other with a double strand specific ligase

1 . A method for generating an amplified nucleic acid portion of a template nucleic acid, which is RNA, the method comprising:
 obtaining said template RNA;   annealing at least one oligonucleotide primer to said template RNA;   annealing at least one oligonucleotide stopper and/or further primer to said template RNA;   elongating the at least one oligonucleotide primer in a template specific manner until the elongating product nucleic acid reaches the position of an annealed oligonucleotide stopper or further primer, whereby the elongation reaction is stopped, wherein in said elongation reaction said optional oligonucleotide stopper is not elongated and/or said further oligonucleotide primer is elongated in a template specific manner;   wherein the elongated product nucleic acid is ligated to the 5′ end of said oligonucleotide stopper or further primer.   
     
     
         2 . A method for generating an amplified nucleic acid portion of a template nucleic acid, the method comprising:
 obtaining said template nucleic acid;   annealing at least one oligonucleotide primer to said template nucleic acid;   annealing at least one oligonucleotide stopper to said template nucleic acid;   elongating the at least one oligonucleotide primer in a template specific manner until the elongating product nucleic acid reaches the position of an annealed oligonucleotide stopper, whereby the elongation reaction is stopped, wherein in said elongation reaction said oligonucleotide stopper is not elongated; and   wherein the elongated product nucleic acid is ligated to the 5′ end of said oligonucleotide stopper.   
     
     
         3 . The method of  claim 1 , wherein the oligonucleotide stopper is hybridized to at least one further oligonucleotide primer. 
     
     
         4 . A method of generating an amplified nucleic acid of a template nucleic acid, the method comprising:
 obtaining said template nucleic acid;   annealing a first oligonucleotide primer to said template nucleic acid;   annealing at least one further oligonucleotide primer to said template nucleic acid; and   elongating said first oligonucleotide primer in a template specific manner until the elongating product nucleic acid reaches the position of one of said further oligonucleotide primers, whereby the elongation reaction is stopped, and at least one further oligonucleotide primer is elongated in a template specific manner, wherein the stopped elongated product nucleic acid is ligated to the 5′ end of said further oligonucleotide primer.   
     
     
         5 . The method of  claim 1 , wherein at least one, preferably all, oligonucleotide primer(s) is/are hybridized to a oligonucleotide stopper. 
     
     
         6 . The method according to  claim 1 , wherein the template nucleic acid is RNA or DNA, preferably RNA. 
     
     
         7 . The method according to  claim 1 , further comprising annealing an additional oligonucleotide primer to said template nucleic acid and elongating said additional oligonucleotide primer until the elongating product nucleic acid reaches the position of another oligonucleotide primer or an oligonucleotide stopper, preferably wherein 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more different oligonucleotide primers are used, especially preferred wherein the oligonucleotide primers are random primers. 
     
     
         8 . The method according to  claim 1 , further comprising annealing an additional oligonucleotide stopper to said template nucleic acid and wherein in said elongation reaction said additional oligonucleotide stopper is not elongated, preferably wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more different oligonucleotide stoppers are used, especially preferred wherein the oligonucleotide stoppers are random stoppers. 
     
     
         9 . The method according to  claim 1 , characterized in that the any one of the oligonucleotide stoppers comprises a sequence tag, preferably a uniform sequence tag being attached to more than one of the oligonucleotide stoppers, especially preferred to all of the oligonucleotide stoppers, in particular wherein said sequence tag is attached to the 3′ end of the oligonucleotide stopper. 
     
     
         10 . The method according to  claim 1 , characterized in that any one of the oligonucleotide primers comprises a sequence tag, preferably a uniform sequence tag being attached to more than one of the oligonucleotide primers, especially preferred to all of the oligonucleotide primers, in particular wherein said sequence tag is attached to the 5′ end of the oligonucleotide primer. 
     
     
         11 . The method of  claim 10 , wherein any one of the oligonucleotide stoppers and any one of the oligonucleotide primers comprises a sequence tag each, and preferably wherein the sequence tag of the oligonucleotide primers is at least partially complementary with the sequence the sequence tag of the oligonucleotide stopper thereby enabling hybridization of the oligonucleotide stoppers and the oligonucleotide primers with each other at least in a part of the respective sequence tag. 
     
     
         12 . The method according to  claim 1 , characterized in that said labeling step comprises ligation with a sequence tag attached to said oligonucleotide primers or oligonucleotide stoppers or comprises ligation with said oligonucleotide primers or oligonucleotide stoppers, which comprise a sequence tag. 
     
     
         13 . The method according to  claim 12 , wherein said sequence tag is hybridized to the 5′ end of said oligonucleotide primer or oligonucleotide stopper, preferably wherein said sequence tag is ligated to the elongation product of an upstream primer. 
     
     
         14 . The method according to  claim 1 , characterized in that said labeling step comprises ligation with an oligonucleotide primer or oligonucleotide stopper comprising a sequence tag, preferably wherein said sequence tag is covalently attached to said oligonucleotide primer or oligonucleotide stopper or wherein nucleic acid comprising said sequence tag is hybridized with said oligonucleotide primer or oligonucleotide stopper. 
     
     
         15 . The method according to  claim 14 , wherein said sequence tag is ligated or hybridized to the 3′ end of an oligonucleotide stopper. 
     
     
         16 . The method according to  claim 1 , wherein a nucleic acid with a reverse complement sequence to the tag sequence is added during the annealing or elongation reaction or is hybridized to the sequence tag. 
     
     
         17 . The method according to  claim 16 , wherein the melting temperature of the sequence tag with the nucleic acid with the reverse complement sequence is increased as compared to unmodified nucleic acid by modified nucleotides, preferably selected from one or more LNA nucleotides, 2′ fluoronucleotides or PNA nucleotides. 
     
     
         18 . The method according to  claim 16 , wherein the nucleic acid with the reverse complement sequence is covalently linked to the sequence tag, preferably via a spacer or hairpin loop. 
     
     
         19 . The method according to  claim 9 , further comprising amplifying said elongated products comprising said ligated tag, preferably by PCR using tag specific primers. 
     
     
         20 . The method according to  claim 1 , characterized in that the oligonucleotide primers and/or oligonucleotide stoppers are phosphorylated or adenlyated on the 5′ end. 
     
     
         21 . The method according to  claim 1 , characterized in that the template nucleic acid is immobilized on a solid phase or solid support. 
     
     
         22 . The method according to  claim 1 , further comprising the step of washing elongated product nucleic acids, preferably hybridized to a template nucleic acid, especially hybridized to an immobilized template nucleic acid according to  claim 21 . 
     
     
         23 . The method according to  claim 1 , characterized in that elongation and ligation reactions are performed concurrently in one reaction step. 
     
     
         24 . The method according to  claim 1 , wherein said method is performed by addition by a DNA polymerase and/or a ligase, preferably wherein said DNA polymerase and ligase are added in one reaction mixture with the template nucleic acid. 
     
     
         25 . The method according to  claim 1 , characterized in that said oligonucleotide primer and/or oligonucleotide stopper comprises a nucleotide modification increasing the Tm or stiffens the sugar phosphate backbone of said oligonucleotide, preferably selected from 2′fluoro nucleosides, LNAs, ZNAs, PNAs, or by using intercalators or additives that specifically bind to nucleic acids, such as Ethidium bromide, Sybr Green, preferably intercalators that are specific for RNA:DNA hybrids. 
     
     
         26 . The method according to  claim 25 , characterized in that said oligonucleotide primer and/or oligonucleotide stopper comprises at least one, preferably at least 2, more preferred at least 3 modified nucleotides being selected from G or C, preferably at the 5′ end of the primer sequence. 
     
     
         27 . The method according to  claim 1 , characterized in that a polymerase having nucleotide strand displacement activity is used for the elongation. 
     
     
         28 . The method according to  claim 1 , characterized in that the nucleotide concentration during the polymerization reaction is lower than the template concentration, preferably the molar ratio of nucleotides concentration to template concentration is in the range of 1:33 to 1:3.3. 
     
     
         29 . The method according to  claim 1 , characterized in that actinomycin D is added to the polymerisation reaction in sufficient amounts to avoid second strand synthesis and to reduce strand displacement of the polymerase. 
     
     
         30 . The method according to  claim 1 , o wherein said template nucleic acid is single stranded. 
     
     
         31 . The method according to  claim 1 , wherein the obtained template nucleic acid lacks a complementary strand over at least 30% of its length and/or lacks a complementary strand of at least 100 nucleotides in length, preferably lacks a complementary nucleic acid over its entire length. 
     
     
         32 . (canceled) 
     
     
         33 . A kit for generating amplified nucleic acid portions of a template nucleic acid according to  claim 1  comprising a DNA polymerase, random oligonucleotide primers which comprise a modification that increases the Tm and random oligonucleotide stoppers that are unsuitable for nucleotide extension and comprise a modification that increases the Tm, optionally further one or more of reaction buffers comprising Mn 2+  or Mg 2+ , a ligase, a crowding agent, such as PEG. 
     
     
         34 . A kit for generating amplified nucleic acid portions of a template nucleic acid according to  claim 1 , comprising a DNA polymerase and a ligase, preferably further oligonucleotide primers, oligonucleotide stoppers, a crowding agent, especially PEG, or combinations thereof. 
     
     
         35 . A kit for generating amplified nucleic acid portions of a template nucleic acid containing a) random oligonucleotide primers which comprise a modification that increases the Tm and b) random oligonucleotide stoppers that are unsuitable for nucleotide extension and comprise a modification that increases the Tm. 
     
     
         36 . The kit of  claim 35  further comprising one or more of reaction buffers comprising Mn 2+  or Mg 2+ , a ligase, a crowding agent, such as PEG. 
     
     
         37 . The kit of  claim 35  further comprising a DNA polymerase and/or a ligase.