| 主权项 |
1. A method of selecting nucleic acid samples from a plurality of nucleic acid samples based on one or more desired alleles at one or more polymorphic sites, the method comprising:
(A) setting a user-set value C based on the testing efficiency desired by the user to a value between 0 and 1, but not equal to 0 or 1; (B) calculating for each desired allele a value “d,” wherein “d” is a positive integer determined by the user-set value C and the frequency of the desired allele in a population of individuals; (C) grouping into mathematical bins one or more of said desired alleles, wherein all alleles in a mathematical bin have the same value for “d”; (D) determining a value for the maximum pool size, wherein the maximum pool size is the maximum number of samples in a pool, wherein the value is a number greater than one and is based on technical limitations of performing the steps of the herein method of identifying alleles; (E) selecting one bin from the mathematical bins of alleles determined in step (C) with the proviso that if “d” for the selected bin is greater than the maximum pool size determined in step (D), then “d” for the bin is set to the maximum pool size; (F) collecting aliquots from “d” number of nucleic acid samples of said plurality of nucleic acid samples, and combining said aliquots to form at least one nucleic acid sample pool, wherein each nucleic acid sample pool contains exactly “d” aliquots; (G) (i) performing a reaction in each nucleic acid sample pool to produce reaction products comprising (a) polymorphic site(s) comprising the allele(s) of the selected bin and (b) a source tag identifying each pool, wherein said reaction products are produced using as templates said nucleic acid samples in each said pool;
(ii) if “d” is less than the maximum pool size, then forming at least one pooled pool comprising at least some of the reaction products from a number of pools, said number of pools determined as the result of dividing the maximum pool size by “d”; wherein if said number of pools is not an integer, then said number of pools is set to the closest lesser positive integer;(iii) if “d” is equal to the maximum pool size, then proceeding to step (H) without forming pooled pools; (H) for each of the alleles of the one or more desired alleles, performing a second reaction in the at least one pool or pooled pool using said reaction products comprising said source tag to produce allele-specific second reaction products comprising a marker tag, wherein said marker tag identifies an allele at a polymorphic site; (I) interrogating said marker tags; (J) (i) if all marker tags in a pool or pooled pool are identical and, if said marker tags correspond to the one or more desired alleles, then selecting the nucleic acid samples in said pool or pooled pool as being homozygous for said allele of the one or more desired alleles; or
(ii) if all marker tags in a pool or pooled pool are identical, and if said marker tags do not correspond to the one or more desired alleles, then optionally eliminating from consideration the nucleic acid samples in said pool or pooled pool; or(iii) if all marker tags in a pool or pooled pool are not identical, then:
(a) interrogating the source tags of the second reaction products in said pool or pooled pool to determine which nucleic acid samples were placed into that pool or pooled pool;(b) identifying the marker tags of the second reaction products in said pool or pooled pool; and(c) if said pool or pooled pool comprises a second reaction product with at least one marker tag corresponding to an allele of the one or more desired alleles, then selecting the nucleic acid samples in said pool or pooled pool as containing at least one copy of an allele of the one or more desired alleles. |
