DNA amplificationcan be accomplished at a constant temperature in short time in one step. test results is among the most importantissues in simplification and generalization of genetic testing The mechanism ofthe LAMP reaction is described briefly in Figure (1-13). This method is basedon the use of a specially designed set of four target-specific primers (F3, B3,FIP and BIP), which are able to recognize six distinct sites flanking theamplified DNA sequence (F1c, F2c, F3c sites on the 3¢ side and B1, B2, B3 siteson the 5¢ side (F is for forward and B is for backward directions; the indexletter c is used to distinguish between complementary DNA sequences). One pairof primers, F3 and B3, represents the ordinary, single-domain primers (theyeach recognize one of the six sites and so amplify the entire target region),and these are supplemented by an additional pair of composite, double-domainprimers, FIP and BIP, which recognize two of the six sites each and play themajor role in the LAMP reactions (FIP and BIP primers are used in excess,compared to F3 and B3 primers). The LAMP reaction is considered to progressthrough two steps by DNA polymerase with strand displacement activity: startingstructure producingstep and cycling amplification step.
Production of the starting structurerequires all four primers. At the starting structure-producing step, one of theinner primers (i.e., FIP) works first, thus initiating the replicative DNAsynthesis by Bst DNA polymerase.
Next, the outer primer, F3, takes its place,displacing the newly synthesized DNA strand and releasing the target DNA. Withsimilar performance of BIP and B3, this results in the single-stranded dumbbelllikestarting structure withloops at both ends.This DNA construct is a startingmaterial for the amplification stageof LAMP, which proceeds withthe participation of the two composite primers only. In this stage, thestarting structure is converted to stem–loop DNA by self-primed DNA synthesis,and then FIP hybridizes and primes strand displacement DNA synthesis so thatthe complement of this structure is produced.
It means that the cycle reactionbetween two structure is established.