Rna templated ligation

Abstract

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

1 . A method of detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, said method comprising:
 a) contacting said sample with a padlock probe and allowing said probe to hybridise to the target nucleic acid molecule;   b) subjecting said sample to a ligation reaction using a DNA/RNA ligase to ligate, and thereby circularise, any probe which has hybridised to the target nucleic acid molecule;   c) amplifying the ligated circularised probe from step (b) by rolling circle amplification with a DNA polymerase; and   d) detecting the amplification product from step (c), thereby to detect the target nucleic acid sequence;   wherein the padlock probe is provided in one or more parts, each part having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule such that, optionally after a step of cleaving the hybridised probe and/or extending a 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence; and   wherein said probe comprises at least one ribonucleotide at or near to a ligation site, optionally after said step of cleaving and/or extension, and the ligated circularised probe is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid sequence is a target RNA sequence and wherein the target nucleic acid molecule is a target RNA molecule. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the probe comprises two or more parts, wherein at least two ligation sites are created. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the probe comprises the at least one ribonucleotide at or near a ligatable 3′ end at a ligation site. 
     
     
         5 . The method of  claim 4 , wherein the ligatable 3′ end is created by extension of the 3′ end of the probe or a part thereof hybridised to the target nucleic acid molecule. 
     
     
         6 . The method of any one of  claims 1  to  4 , wherein the probe comprises the at least one ribonucleotide at or near the 3′ end of the probe or a part thereof. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein a 3′ terminal and/or 3′ penultimate nucleotide of the probe or a part thereof is a ribonucleotide. 
     
     
         8 . The method of any one of  claims 1  to  7 , wherein the probe does not comprise a ribonucleotide at a 5′ ligatable end at a ligation site. 
     
     
         9 . The method of any one of  claims 1  to  8 , wherein the 5′ ligatable end of the probe, or a part thereof, is created by cleavage of the probe when it is hybridised to the target molecule. 
     
     
         10 . The method of  claim 9 , wherein a first target-specific target binding site is situated internally of the 5′ end of the probe or probe part and a second target-specific binding site is situated at the 3′ end of the probe or probe part, such that upon hybridisation of the probe or probe parts to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the first target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap which is removed by cleavage thereby to create a 5′ ligatable end. 
     
     
         11 . The method of  claim 10 , wherein one or more nucleotides at the 3′ end of the additional sequence which forms the 5′ flap are complementary to cognate nucleotides in the target nucleic acid molecule, wherein said nucleotides are not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe or probe part. 
     
     
         12 . The method of  claim 10  or  11 , wherein the additional sequence comprises one or more ribonucleotides. 
     
     
         13 . The method of  claim 12 , wherein one or more nucleotides at the 3′ end of the additional sequence are ribonucleotides. 
     
     
         14 . The method of any one of  claims 10  to  13 , wherein the 3′-most nucleotide of the additional sequence is a ribonucleotide. 
     
     
         15 . The method of any one of  claims 1  to  14 , wherein said probe is single circularisable oligonucleotide comprising a first target-specific binding site at or internally to the 5′ end of the probe and a second target-specific binding site at the 3′ end of the probe, wherein the first and second target-specific binding sites form or constitute the 5′ and 3′ ligatable ends of the probe respectively. 
     
     
         16 . The method of any one of  claims 9  to  15 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific target binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the additional sequence is a ribonucleotide which is complementary to the cognate nucleotide in the target nucleic acid molecule but is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe, wherein said additional sequence is removed by cleavage thereby to create the 5′ ligatable end of the probe. 
     
     
         17 . The method of any one of  claims 9  to  16 , wherein cleavage is performed by an enzyme having 5′ nuclease activity or by a structure-specific nuclease. 
     
     
         18 . The method of  claim 17 , wherein said enzyme is a DNA polymerase enzyme selected from  Thermus aquaticus, Thermus thermophilus  or  Thermus flavus  polymerase. 
     
     
         19 . The method of any one of  claims 9  to  18 , wherein cleavage is performed by a FLAP endonuclease. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the ligated probe comprises no more than 2 consecutive ribonucleotides. 
     
     
         21 . The method of any one of  claims 1  to  20 , wherein the DNA polymerase is Bst polymerase or Phi29 DNA polymerase. 
     
     
         22 . The method of any one of  claims 1  to  21 , wherein the ligase is T4 RNA ligase 1, T4 RNA ligase 2 or PBCV-1 DNA ligase. 
     
     
         23 . The method of any one of  claims 1  to  22 , wherein the method is for the detection of a variant base in a target nucleic acid molecule. 
     
     
         24 . The method of  claim 23 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the probe is a ribonucleotide and wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein when the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the variant base the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe and said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe are not complementary to the variant base, the nucleotide at the 3′ end of the additional sequence is removed by cleavage and the nucleotide at the 3′ end of the probe is not hybridised to the target nucleic acid molecule, thereby preventing ligation. 
     
     
         25 . The method of  claim 23 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the probe comprises an additional sequence 5′ to the first target binding site which is not hybridised to the target nucleic acid molecule, wherein the nucleotide at the 3′ end of the probe is a ribonucleotide, and wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base and wherein the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe, wherein when the nucleotide at the 5′ end of the first target-specific binding site is complementary to the variant base, said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 5′ end of the first target-specific binding site is not complementary to the variant base, said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation. 
     
     
         26 . A chimeric DNA-RNA padlock probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein:
 (i) the probe comprises one or more parts each having at least one target specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule such that, optionally after a step of cleaving the hybridised probe and/or extending a 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create one or more ligation sites at or adjacent to the target nucleic acid sequence, wherein ligation at said ligation sites circularises the probe;   (ii) the probe comprises at least one ribonucleotide at or near to a ligation site; and   (iii) the probe when ligated to form a circle is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides.   
     
     
         27 . The chimeric DNA-RNA padlock probe of  claim 26 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific target binding site situated at or internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, and wherein:
 (i) the second target specific binding site comprises one or more ribonucleotides; 
 (ii) where the first target specific binding site is internal to the 5′ end of the probe, the probe comprises an additional sequence 5′ to the first target specific binding site, such that when the probe is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of the probe to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; and 
 (iii) the probe when ligated to form a circle is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides. 
 
     
     
         28 . The chimeric DNA-RNA padlock probe of  claim 26 , wherein the probe comprises two or more parts, the first part being a backbone oligonucleotide comprising a first target-specific target binding site situated at or internally of its 5′ end and a second target-specific binding site situated at its 3′ end, and one or more gap oligonucleotides which each comprise a target-specific binding site complementary to and capable of hybridising to the target nucleic acid molecule in between the first and second target-specific binding sites of the backbone oligonucleotide, and wherein:
 (i) the second target specific binding site of the backbone oligonucleotide and/or at the target-specific binding site of at least one gap oligonucleotide comprises one or more ribonucleotides at or near the 3′ end thereof; 
 (ii) where the first target specific binding site is internal to the 5′ end of the backbone oligonucleotide, the backbone oligonucleotide comprises an additional sequence 5′ to the first target specific binding site, such that when the backbone oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of a gap oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; 
 (iii) one or more gap oligonucleotides optionally comprise an additional sequence 5′ to the target-specific binding site, such that when the gap oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of another gap oligonucleotide or to the 3′ end of the backbone oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides; and 
 (iv) the backbone and gap oligonucleotides when ligated form a circle which is composed primarily of DNA and comprises no more than 4 consecutive ribonucleotides. 
 
     
     
         29 . The probe of  claim 27  or  28 , wherein the probe or backbone oligonucleotide and/or one or more gap oligonucleotides comprises an additional sequence 5′ to the first target-specific binding site or to a target-binding site of a gap oligonucleotide, wherein the nucleotide at the 3′ end of any additional sequence which forms a 5′ flap is complementary to the same cognate nucleotide in the target nucleic acid molecule as the nucleotide at the 3′ end of the probe, or at the 3′ end of the backbone and/or one or more gap oligonucleotides. 
     
     
         30 . The probe of  claim 29 , wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein:
 (i) the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides and the nucleotide at the 3′ end of the additional sequence are complementary to a variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or   (ii) the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides are not complementary to the variant base, such that the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotides is not capable of hybridising to the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3′ end of the additional sequence may be removed by cleavage.   
     
     
         31 . The probe of  claim 29 , wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein:
 (i) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a backbone or gap oligonucleotide is complementary to the variant base, the nucleotide at the 3′ end of the probe or backbone and/or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or   (ii) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a backbone or gap oligonucleotide is not complementary to the variant base, and said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.   
     
     
         32 . A set of two or more probes as defined in  claim 30  or  claim 31 , wherein each probe in said set of probes comprises a different nucleotide at a position in the 3′ end of the probe and/or backbone or gap oligonucleotide and first target binding site or additional sequence complementary to the variant base. 
     
     
         33 . The set of probes of  claim 32 , comprising three or four probes, wherein each probe comprises a different nucleotide at said position. 
     
     
         34 . A method of detecting a target nucleic acid sequence in a target nucleic acid molecule in a sample, said method comprising:
 a) contacting said sample with a ligatable probe and allowing said probe to hybridise to the target nucleic acid molecule;   b) subjecting said sample to a ligation reaction using a DNA/RNA ligase to ligate any probe which has hybridised to the target nucleic molecule;   c) amplifying the ligated probe from step (b) with a DNA polymerase; and   d) detecting the amplification product from step (c), thereby to detect the target nucleic acid sequence;   
       wherein each probe is provided in one or more parts each having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequence and hybridises to the target nucleic acid molecule, wherein one target-specific binding site of the probe or at least one target-specific binding site of a probe part is situated internally of the 5′ end of the probe or probe part such that upon hybridisation of the probe or probe part to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap, wherein one or more of the nucleotides at the 3′ end of the 5′ additional sequence is a ribonucleotide, and wherein after a step of cleaving the hybridised probe to remove the 5′ flap, and optionally after a step of extending the 3′ end thereof using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence. 
     
     
         35 . The method of  claim 34 , wherein the probe comprises two or more parts, and wherein at least two ligation sites are created. 
     
     
         36 . The method of  claim 34  or  35 , wherein the ligatable 3′ end is created by extension of the 3′ end of the probe or a part thereof hybridised to the target nucleic acid molecule. 
     
     
         37 . The method of any one of  claims 34  to  36 , wherein one or more nucleotides at the 3′ end of the additional sequence which forms the 5′ flap are complementary to cognate nucleotides in the target nucleic acid molecule, wherein said nucleotides are not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe or probe part. 
     
     
         38 . The method of any one of  claims 34  to  37 , wherein the probe is a padlock probe comprising one or more parts which are ligated together to form a circle, optionally after an extension step. 
     
     
         39 . The method of  claim 38 , wherein said probe is a single circularisable oligonucleotide comprising first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site at the 3′ end of the probe, wherein the first and second target-specific binding sites form or constitute the 5′ and 3′ ligatable ends of the probe respectively. 
     
     
         40 . The method of any one of  claims 34  to  39 , wherein the probe is a single circularisable oligonucleotide comprising a first target specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, such that upon hybridisation of the probe to the target nucleic acid molecule the nucleotide at the 3′ end of the additional sequence is complementary to the cognate nucleotide in the target nucleic acid molecule but is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ ligatable end of the probe. 
     
     
         41 . The method of any one of  claims 34  to  40 , wherein cleavage is performed by an enzyme having 5′ nuclease activity or by a structure-specific nuclease. 
     
     
         42 . The method of  claim 41 , wherein said enzyme is a DNA polymerase enzyme selected from  Thermus aquaticus, Thermus thermophilus  or  Thermus flavus  polymerase. 
     
     
         43 . The method of any one of  claims 34  to  40 , wherein cleavage is performed by a FLAP endonuclease. 
     
     
         44 . The method of any one of  claims 34  to  43 , wherein the ligated probe comprises no more than 4 consecutive ribonucleotides, preferably wherein the ligated probe comprises no more than 3, or no more than 2 consecutive ribonucleotides. 
     
     
         45 . The method of any one of  claims 34  to  44 , wherein the ligated probe does not comprise a ribonucleotide at or near to a ligation site. 
     
     
         46 . The method of any one of  claims 34  to  45 , wherein where the probe is circularised by ligation, the amplification is rolling circle amplification. 
     
     
         47 . The method of any one of  claims 34  to  46 , wherein amplification is PCR or a variant thereof, SDA, HDA, LAMP or SMAP. 
     
     
         48 . The method of any one of  claims 34  to  47 , wherein the DNA polymerase is Bst polymerase or Phi29 DNA polymerase. 
     
     
         49 . The method of any one of  claims 34  to  48 , wherein the ligase is T4 RNA ligase 1, T4 RNA ligase 2 or PBCV-1 DNA ligase. 
     
     
         50 . The method of any one of  claims 34  to  49 , wherein the method is for the detection of a variant base in a target nucleic acid molecule. 
     
     
         51 . The method of  claim 50 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at 3′ end of the probe, wherein the nucleotide at the 3′ end of the probe and the 3′ end of the additional sequence are complementary to the same nucleotide, wherein when the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the variant base the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe and the additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 3′ end of the additional sequence and the nucleotide at the 3′ end of the probe are not complementary to the variant base, the nucleotide at the 3′ end of the additional sequence is removed by cleavage and the nucleotide at the 3′ end of the probe is not hybridised to the target nucleic acid molecule, thereby preventing ligation. 
     
     
         52 . The method of  claim 51 , wherein the probe is a single circularisable oligonucleotide comprising a first target-specific binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the same nucleotide, wherein the nucleotide at the 3′ end of the probe and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base and wherein the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the nucleotide at the 3′ end of the probe, wherein when the nucleotide at the 5′ end of the first target-specific binding site is complementary to the variant base, said additional sequence is removed by cleavage thereby to generate the 5′ ligatable end of the probe, and wherein when the nucleotide at the 5′ end of the first target-specific binding site is not complementary to the variant base, said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation. 
     
     
         53 . A chimeric DNA-RNA probe capable of binding to and detecting a target nucleic acid sequence in a target nucleic acid molecule, wherein the probe comprises one or more parts each having at least one target-specific binding site which is complementary to a cognate probe-binding site at or adjacent to the target nucleic acid sequences and hybridises thereto, wherein one target-specific binding site of the probe or at least one target-specific binding site of a probe part is situated internally of the 5′ end of the probe or probe part such that upon hybridisation of the probe or probe part to the target nucleic acid molecule the probe or probe part comprises an additional sequence 5′ to the target-specific binding site which is not hybridised to the target nucleic acid molecule and forms a 5′ flap, wherein one or more of the nucleotides at the 3′ end of the 5′ additional sequence is a ribonucleotide, and wherein after a step of cleaving the hybridised probe to remove the 5′ flap, and optionally after a step of extending the 3′ end of the probe or a probe part using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create a ligation site at or adjacent to the target nucleic acid sequence. 
     
     
         54 . The chimeric DNA-RNA probe of  claim 53 , wherein the probe is a padlock probe, wherein the probe hybridises to the target nucleic acid molecule such that after cleavage of the additional sequence and optionally after a step of extending the 3′ end of the probe or a probe part using the target nucleic acid molecule as a template, ligatable ends of the probe or probe parts are juxtaposed for ligation to each other using the target nucleic acid molecule as a ligation template, to create one or more ligation sites at or adjacent to the target nucleic acid molecule and wherein ligation at said ligation sites circularises the probe. 
     
     
         55 . The chimeric DNA-RNA probe of  claim 54 , wherein the probe is a single circularisable oligonucleotide comprising at first target-specific target binding site situated internally of the 5′ end of the probe and a second target-specific binding site situated at the 3′ end of the probe. 
     
     
         56 . The chimeric DNA-RNA probe of  claim 55 , wherein the probe comprises two or more parts, the first part being a backbone oligonucleotide comprising a first target-specific target binding site situated internally of its 5′ end and a second target-specific binding site situated at its 3′ end, and one or more gap oligonucleotides which each comprise a target-specific binding site complementary to and capable of hybridising to the target nucleic acid molecule in between the first and second target-specific binding sites of the backbone oligonucleotide, and wherein one or more gap oligonucleotides optionally comprise an additional sequence 5′ to the target-specific binding site, such that when the gap oligonucleotide is hybridised to the target nucleic acid molecule the additional sequence forms a 5′ flap which is not hybridised to the target nucleic acid molecule, and which may be removed by cleavage to generate a ligatable 5′ end which may be ligated to the 3′ end of another gap oligonucleotide or to the 3′ end of the backbone oligonucleotide to circularise the probe, wherein the additional sequence may optionally contain one or more ribonucleotides. 
     
     
         57 . The chimeric DNA-RNA probe of any one of  claims 53  to  56 , wherein the nucleotide at the 3′ end of any additional sequence which forms a 5′ flap is complementary to the same cognate nucleotide in the target nucleic acid molecule as the nucleotide at the 3′ end of the probe or at the 3′ end of the backbone and/or one or more gap oligonucleotides. 
     
     
         58 . The probe of  claim 57 , wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein:
 (i) the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to a variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or probe part or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or   (ii) the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are not complementary to the variant base, such that the nucleotide at the 3′ end of the probe or probe part or backbone and/or gap oligonucleotides is not capable of hybridising to the target nucleic acid molecule, thereby preventing ligation, and the nucleotide at the 3′ end of the additional sequence may also be removed by cleavage.   
     
     
         59 . The probe of  claim 57 , wherein the probe is for detecting a variant base in a target nucleic acid molecule, and wherein:
 (i) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a probe part or backbone or gap oligonucleotide is complementary to the variant base, the nucleotide at the 3′ end of the probe or probe part or backbone or gap oligonucleotide and the nucleotide at the 3′ end of the additional sequence are complementary to the nucleotide at the position 3′ to the variant base, and the nucleotide at the 3′ end of the additional sequence is not capable of hybridising to the target nucleic acid molecule simultaneously with the 3′ end of the probe or backbone or gap oligonucleotide, such that said additional sequence may be removed by cleavage to generate a 5′ ligatable end of the probe; or   (ii) the nucleotide at the 5′ end of the first target-specific binding site or at the 5′ end of the target-specific binding site of a probe part or a backbone or gap oligonucleotide is not complementary to the variant base, and said nucleotide is also removed by cleavage, thereby generating a gap between the 5′ and 3′ ligatable ends and preventing ligation.   
     
     
         60 . A set of two or more probes as defined in  claim 58  or  59 , wherein each probe in said set comprises a different nucleotide at a position in the 3′ end of the probe or probe part or backbone or gap oligonucleotide and first target binding site or additional sequence complementary to the variant base. 
     
     
         61 . The set of probes of  claim 60 , comprising three or four probes, wherein each probe comprises a different nucleotide at said position. 
     
     
         62 . A method of synthesising a DNA molecule by rolling circle amplification, said method comprising contacting a circular template nucleic acid molecule comprising at least one ribonucleotide and no more than four consecutive ribonucleotides with a Phi29 DNA polymerase, and generating a DNA molecule comprising multiple tandem repeats of the reverse complement sequence to the template nucleic acid molecule, wherein the sequence of the Phi29 DNA polymerase has not been modified to increase RT activity. 
     
     
         63 . The method of  claim 62 , wherein the template nucleic acid molecule comprises no more than 3 consecutive ribonucleotides. 
     
     
         64 . The method of  claim 62  or  63 , wherein the template nucleic acid molecule comprises no more than 2 consecutive ribonucleotides. 
     
     
         65 . The method of any one of  claims 62  to  64 , wherein the template nucleic acid molecule comprises 2 or more consecutive ribonucleotides. 
     
     
         66 . The method of any one of  claims 62  to  65 , wherein the template nucleic acid molecule comprises one or more single ribonucleotides. 
     
     
         67 . The method of any one of  claims 62  to  66 , wherein the template nucleic acid molecule comprises ribonucleotides within a single site within the template nucleic acid molecule. 
     
     
         68 . The method of any one of  claims 62  to  67 , wherein the template nucleic acid molecule comprises 1 ribonucleotide. 
     
     
         69 . The method of any one of  claims 62  to  66 , wherein the template nucleic acid molecule contains ribonucleotides at two or more separate sites, and comprises one or more deoxyribonucleotides between each site. 
     
     
         70 . The method of  claim 69 , wherein the template nucleic acid molecule contains ribonucleotides at two or more separate sites, wherein two sites are separated by a single deoxyribonucleotide. 
     
     
         71 . The method of  claim 65 , wherein the consecutive ribonucleotides are homogeneous. 
     
     
         72 . The method of any one of  claims 62  to  70 , wherein at least one of the ribonucleotides is a pyrimidine ribonucleotide, or the method of  claim 71  wherein the consecutive ribonucleotides are pyrimidine ribonucleotides. 
     
     
         73 . The method of  claim 69  or  70 , wherein each site contains ribonucleotides as defined in any one of  claim 63  to  66  or  71 . 
     
     
         74 . The method of any one of  claims 62  to  73 , wherein the ribonucleotide is not a chemical modification, analogue or derivative of a ribonucleotide. 
     
     
         75 . The method of any one of  claims 62  to  74 , wherein the circular nucleic acid molecule is formed following the ligation of a padlock probe provided in one or more parts using a target nucleic acid molecule as a template, wherein said ligation comprises ligation of ligatable ends of a probe or probe parts to create a ligation site. 
     
     
         76 . Use of Phi29 DNA polymerase as a reverse transcriptase enzyme wherein the sequence of the Phi29 polymerase has not been modified to increase RT activity.