LOCALISED RCA-BASED AMPLIFICATION METHOD USING A PADLOCK-PROBE

摘要

The present invention provides a method for performing a localised RCA reaction comprising at least two rounds of RCA, wherein the product of a second RCA reaction is attached, and hence localised, to a product of a first RCA reaction, said method comprising: (a) providing a concatemeric first RCA product comprising repeated monomers; (b) directly or indirectly hybridising to monomers of said first RCA product a circularisable oligonucleotide comprising target-complementary 3′ and 5′ end regions such that the 3′ and 5′ ends of said oligonucleotide hybridise in juxtaposition for ligation directly or indirectly to each other, wherein the target is a sequence in a monomer of said first RCA product or an intermediate molecule hybridised thereto, and wherein the target-complementary end regions of said circularisable oligonucleotide are 6 to 16 nucleotides in length; (c) directly or indirectly ligating the ends of said circularisable oligonucleotide to circularise the oligonucleotide, thereby to provide a template for a second RCA reaction, wherein when said ends are indirectly ligated (i) either a gap oligonucleotide is provided which hybridises to the monomers of the first RCA product in between the 3′ and 5′ ends of the circularisable oligonucleotide such that it may be ligated to the respective ends, or the hybridised 3′ end of the circularisable oligonucleotide is extended by a polymerase such that the extended 3′ end may be ligated to the hybridised 5′ end, and (ii) the total length of the region of the second RCA template directly or indirectly hybridised to the monomers is no longer than 32 nucleotides in length; and (d) performing a second RCA reaction using said second RCA template of (c) and a primer for said second RCA, to form a second RCA product, wherein in said second RCA reaction the second RCA template remains attached to the first RCA product, and thereby the second RCA product is attached to the first RCA product.

主权项

1. A method for performing a localised RCA reaction comprising at least two rounds of RCA, wherein the product of a second RCA reaction is attached, and hence localised, to a product of a first RCA reaction, said method comprising:
(a) providing a concatemeric first RCA product comprising repeated monomers; (b) directly or indirectly hybridising to monomers of said first RCA product a circularisable oligonucleotide comprising target-complementary 3′ and 5′ end regions such that the 3′ and 5′ ends of said oligonucleotide hybridise in juxtaposition for ligation directly or indirectly to each other, wherein the target is a sequence in a monomer of said first RCA product or an intermediate molecule hybridised thereto, and wherein the target-complementary end regions of said circularisable oligonucleotide are 6 to 16 nucleotides in length; (c) directly or indirectly ligating the ends of said circularisable oligonucleotide to circularise the oligonucleotide, thereby to provide a template for a second RCA reaction, wherein when said ends are indirectly ligated (i) either a gap oligonucleotide is provided which hybridises to the monomers of the first RCA product in between the 3′ and 5′ ends of the circularisable oligonucleotide such that it may be ligated to the respective ends, or the hybridised 3′ end of the circularisable oligonucleotide is extended by a polymerase such that the extended 3′ end may be ligated to the hybridised 5′ end, and (ii) the total length of the region of the second RCA template directly or indirectly hybridised to the monomers is no longer than 32 nucleotides in length; and (d) performing a second RCA reaction using said second RCA template of (c) and a primer for said second RCA, to form a second RCA product, wherein in said second RCA reaction the second RCA template remains attached to the first RCA product, and thereby the second RCA product is attached to the first RCA product.