METHOD FOR ASSEMBLING MULTIPLE TARGET LOCI TO A SINGLE NUCLEIC ACID SEQUENCE

摘要

The present invention relates to a method for assembling multiple target loci to a single assembled nucleic acid sequence, and to a method for simultaneously detecting multiple target loci using said assembly method. The method of the present invention involves assembling the multiple target loci to a single assembled nucleic acid sequence through two polymerase chain reactions (PCRs), i.e. a primary PCR and a secondary PCR. More particularly, a pair of primers (a forward primer and an inverse primer) for a primary amplification consist of a target-specific sequence (a target hybridization nucleotide sequence) and a 5'-flanking assembly spacer sequence (an overlapping sequence). In addition, a primary amplified product amplified by means of the pair of primers for a primary amplification is assembled to a single shortened nucleic acid sequence in a convenient and easy manner through a set of primers for a secondary amplification, thus enabling the simultaneous detection of multiple target loci. Accordingly, the method and a kit of the present invention may simultaneously detect and analyze multiple variabilities in DNA sequences of a sample (preferably human blood), thus not only remarkably reducing sequencing costs for detecting variabilities, but also providing a critical approach and means for achieving the concept of customized medicine.