Patent ID: 6225064
Filing Date: 2001-05-01
Classification: C12Q

Abstract:
A method for assaying DNA fragments in a mixture comprising:(1) ligating oligomers having known different nucleotide sequences to the 3' termini of DNA fragments in each of the DNA fragment groups, the DNA fragments in each of the DNA fragment groups being prepared, respectively, from each of sample DNAs, and each of the oligomers being preselected corresponding to each of the DNA fragment groups;(2) mixing, in one receptacle, the DNA fragments ligated with the oligomers in the DNA fragment groups;(3) performing a polymerase chain reaction to obtain amplified DNA fragments, in the one receptacle, simultaneously, using the mixture of DNA fragments ligated with the oligomers as templates, and using primers each being complementary to each of the oligomers being ligated to the DNA fragments in each of the DNA fragment groups, and each of the primers being preselected corresponding to each of the DNA fragment groups; and(4) detecting the amplified DNA fragments; wherein:the primers comprise plural module sequences;aligning orders of the plural module sequences of the primers are different from one another;each of the aligning orders of each the plural module sequences is preselected corresponding to each of the DNA fragment groups;each of the plural module sequences is composed of 4 to 6 nucleotides and has the same nucleotide species at the 3' terminus and 5' terminus thereof;the plural module sequences have known different nucleotide sequences, respectively;the primers have the same length, melting temperature, and composition of nucleotides;the primers have known different nucleotide sequences, respectively; andthe aligning orders of the plural module sequences are obtained by replacing the aligning orders of two module sequences which have the same nucleotide species at the 3' terminus and 5' terminus thereof; and wherein:the primers are labeled with different fluorophores being preselected corresponding to each of the DNA fragment groups; andthe amplified DNA fragments are labeled with the different fluorophores corresponding to each of the DNA fragment group.