| CPC G16C 20/50 (2019.02) [C12N 15/09 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6876 (2013.01); G16B 25/20 (2019.02); G16B 30/00 (2019.02); G16C 20/20 (2019.02); G16C 20/60 (2019.02); G16C 20/90 (2019.02); C12Q 1/68 (2013.01)] | 10 Claims |
|
1. A method for designing primer pairs for multiplex PCR performed by CPU and an operation program operating the CPU, according to a priority k, the primer pairs PCR amplifying n candidate amplification regions on same chromosomal DNA, and the priority k being assigned to each of the n candidate amplification regions, where k is an integer satisfying 1≤k≤n, and n is an integer satisfying n>3,
the method including: a step of selecting the n candidate amplification regions on same chromosomal DNA;
a step of obtaining coordinate information and identification information, the coordinate information being a coordinate value r of each of the selected n candidate amplification regions, and the identification information being an identification name R of each of the selected n candidate amplification regions that are assigned identification numbers in an increasing order of the coordinate value r of the n candidate amplification regions;
a first priority setting step of, using the coordinate information and the identification information of the n candidate amplification regions, searching for a minimum coordinate value rmin and a maximum coordinate value rmax and setting a candidate amplification region Rmin having the minimum coordinate value Imin as a candidate amplification region Rmin(k=1) having a first priority (k=1) or a candidate amplification region Rmax having the maximum coordinate value rmax as a candidate amplification region Rmax(k=1) having a first priority (k=1);
a second priority setting step of setting the candidate amplification region Rmin having the minimum coordinate value rmin that was not set as having the first priority in the first priority setting step as a candidate amplification region Rmin(k=2) having a second priority (k=2) or the candidate amplification region Rmax having the maximum coordinate value Imax that was not set as having the first priority in the first priority setting step as a candidate amplification region Rmax(k=2) having a second priority (k=2); and
a k-th priority setting step of using the identification information, coordinate information, and priority information of the n candidate amplification regions, searching for a candidate amplification region Ri and a candidate amplification region Rj satisfying a condition that a candidate amplification region R(k=i) has i-th priority (k=i) and a coordinate value ri while a candidate amplification region R(k=j) has j-th priority (k j) and a coordinate value rj, and that no candidate amplification region assigned a priority is present but at least one candidate amplification region yet to be assigned a priority is present between the candidate amplification region Ri and the candidate amplification region Rj,
then calculating a coordinate value ri-j of a midpoint between the candidate amplification region Ri and the candidate amplification region Rj in accordance with ri-j=(ri+rj)/2, and
further searching for and setting a candidate amplification region having a coordinate value closest to the coordinate value ri-j of the midpoint or closest to the midpoint as a candidate amplification region R(k) assigned k-th priority,
the k-th priority setting step being repeated until all of the n candidate amplification regions are each assigned a priority;
a step of selecting a predetermined number of candidate amplification regions from the n candidate amplification regions in order from the highest priority as amplification purpose regions and designing at least one primer pair for each of the selected amplification purpose regions;
where i and j satisfy 1≤i≤k−1, 1≤j≤k−1, and i≠j, ri and rj satisfy rmin≤ri≤rmax, rmin≤rj≤rmax, and ri≠rj; and
using the at least one primer pair in multiplex PCR analysis.
|