PROTOCOLS AND KITS FOR MULTIPLEX AMPLIFICATION AND NGS-SPECIFIC TAGGING
Present invention concerns amplification protocols and hairpin-harbouring primer pairs suitable for multiplex applications, preferably in closed automated systems. In particular, presented herein protocols allow one-tube multiplex amplification of a potentially large amount of targets in a manner that allows to readily tag the products of said amplification with indexes, unique molecular identifiers (UMIs), or other NGS-related tags compatible with different NGS strategies and platforms.
1 . A method of thermocycling comprising providing at least two primer pairs in a mix, the first primer pair being a target-specific primer pair and the second pair being a universal primer pair, and subjecting the mix to a thermocycling protocol,
the method characterised in that
the target-specific primer pair comprises a target-specific primer comprising
a target-specific sequence having melting temperature T m-ts and
at a 5′-end of said target specific sequence a first anisotropical hairpin defined as having a first loop and a first anisotropical stem comprising at least one G-T or G-U pairing and having melting temperature T m-as1 ,
wherein T m-as1 is at least 8° C. higher than T m-ts ;
and in that
the universal primer pair comprises a universal primer comprising a second anisotropical hairpin defined as having a second loop and a second anisotropical stem comprising at least one G-T or G-U pairing, having melting temperature T m-as2 and having at least 90% sequence similarity to the first anisotropical stem.
2 . The method according to claim 1 , wherein the thermocycling protocol comprises an annealing step at a temperature T a being at least 6° C. lower than T m-as1 , and preferably being not more than 2° C. different from T m-ts .
3 . The method according to claim 2 , wherein the temperature difference between T a and T m-ts is not more than 2° C., preferably not more than 1° C., most preferably wherein T a is equal to T m-ts .
4 . The method according to claim 3 , wherein any of T m-ts and/or T a is at least 8° C., preferably °9 C, most preferably 10° C. lower than T m-as1 and T m-as2 .
5 . The method according to claim 4 , wherein any of T m-ts and/or T a is between 52-66° C., preferably 54-64° C., more preferably is between 56-62° C., most preferably is between around 58-68° C.
6 . The method according to claim 4 , wherein the second anisotropical stem has at least 95% sequence similarity to the first anisotropical stem, preferably being 98% sequence similarity, most preferably being identical to the first anisotropical stem, or wherein the difference between T m-as1 and T m-as2 is not more than 3° C., preferably not more than 1° C., most preferably T m-as1 is equal to a T m-as2 .
7 . The method according to claim 6 , wherein T m-as1 and preferably T m-as2 are at least 70° C. or higher.
8 . The method according to claim 2 , wherein the thermocycling protocol further comprises an annealing step at a temperature T a2 , wherein T a2 >T a .
9 . The method according to claim 1 , wherein the concentration of the target-specific primer pair is at least 5 times lower than the concentration of the universal primer pair, preferably being at least 8 times lower, most preferably being at least 10 times lower.
10 . The method according to claim 1 , wherein the PCR mix comprises at least two, preferably more target-specific primer pairs.
11 . The method according to claim 1 , wherein the target-specific primer further comprises a unique molecular identifier (UMI) sequence at the 5′-end of the gene-specific sequence.
12 . The method according to claim 1 , wherein the universal primer pair comprises an index or another Next Generation Sequencing (NGS)-strategy-specific barcode sequence.
13 . The method according to claim 12 , wherein the method results in NGS-ready amplicons.
14 . A kit comprising a target-specific primer pair and a universal primer pair,
the kit characterized in that
the target-specific primer pair comprises a target-specific primer comprising
a target-specific sequence having melting temperature T m-ts and
at a 5′-end of said target specific sequence a first anisotropical hairpin defined as having a first loop and a first anisotropical stem comprising at least one G-T or G-U pairing and having melting temperature T m-as1 ,
wherein T m-as1 is at least 8° C. higher than T m-ts ;
and in that
the universal primer pair comprises a universal primer comprising a second anisotropical hairpin defined as having a second loop and a second anisotropical stem comprising at least one G-T or G-U pairing, having melting temperature T m-as2 and having at least 90% sequence similarity to the first anisotropical stem.
15 . The kit according to claim 14 , wherein the kit comprises a cartridge engageable with an automated platform.