High-density Xuorescently labeled rolling-circle amplicons for DNA diagnostics

Fluorescent labeling of nucleic acids with high density is very important for a wide area of DNAand RNAbased diagnostics [1–5], but it still remains a challenge. Indeed, chemical synthesis of oligonucleotides with multiple Xuorophores is an elaborate and expensive procedure [6–8]. Branched probes tagged with numerous Xuorophores can alleviate this problem [2,9], but special complex assemblies are required in that case. Enzymatic synthesis of high-density labeled polynucleotides from Xuorescently modiWed monomers by DNA polymerases is another alternative. However, despite some progress in this area, there are certain diYculties in enzymatically generating long Xuorescent products using procedures based on primer extension [3,4,10–13]. Rolling-circle ampliWcation (RCA),2 which is isothermal DNA polymerase-driven cyclic replication of small circular DNA probes, is often used as an eYcient method to detect various analytes [14–16]. It was assumed that the RCA mechanism is diVerent from that