METHOD FOR ASSEMBLING MULTIPLE TARGET LOCI TO SINGLE NUCLEIC ACID SEQUENCE

摘要

A method for assembling multiple target loci to a single assembled nucleic acid sequence involves assembling the multiple target loci to a single assembled nucleic acid sequence through two polymerase chain reactions (PCRs). A pair of primers for a primary amplification include a target-specific sequence and a 5′-flanking assembly spacer sequence. A primary amplified product amplified by 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, thus remarkably reducing sequencing costs for detecting variabilities, and providing a critical approach and means for achieving the concept of customized medicine.

主权项

1. A method for assembling multiple target loci into a single shortened nucleic acid sequence, comprising:
(a) obtaining a target nucleic acid molecule including multiple target loci including at least two target loci on one molecule thereof; (b) obtaining primary amplification products by primary amplification of the target nucleic molecule using a primary amplification primer set including at least two primer pairs for being hybridized with upstream and downstream regions of the at least two target loci and amplifying flanking regions of the at least two target loci, wherein the at least two primer pairs each having a forward primer and a reverse primer and the at least two primer pairs include a first primer pair for amplifying a first target locus which is located relatively in the 5′ direction and a second primer pair for amplifying a second target locus which is located in the 3′ direction of the first target locus; wherein a reverse primer of the first primer pair includes (i) a target hybridization nucleotide sequence that is complementary to a downstream region of the first target locus and (ii) an overlapping sequence that is non-complementary to the target nucleic acid molecule but complementary to a forward primer of the second primer pair; and wherein the forward primer of the second primer pair includes (i) a target hybridization nucleotide sequence that is complementary to an upstream region of the second target locus and (ii) an overlapping sequence that is non-complementary to the target nucleic acid molecule but complementary to the reverse primer of the first primer pair; and (c) obtaining secondary amplification products by secondary amplification using a secondary amplification primer set and the primary amplification products, the secondary amplification primer set including a primer that is complementary to a 5′ end region formed when the primary amplification products are arranged in the 5′ to 3′ direction and a primer that is complementary to a 3′ end region of the sequence, wherein the secondary amplification products constitute a nucleic acid sequence in which the at least two target loci are located adjacent to each other, the nucleic acid being extended to have a greater length than the target nucleic acid molecule used in step (a).