Methods of identifying genetic markers in the human cystic fibrosis transmembrane conductance regulator (CFTR) gene
The present invention relates to the field of genetic screening. More specifically, the described embodiments concern methods to screen multiple samples, in a single assay, for the presence or absence of mutations or polymorphisms in a plurality of genes. Approaches to screen for the presence or absence of mutations that are associated with cystic fibrosis and approaches to design primers that generate extension products that facilitate the resolution of multiple extension products in a single lane of a gel or in a single run on a column are also provided.
1. A method of identifying the presence or absence of genetic markers in the human cystic fibrosis transmembrane conductance regulator (CFTR) gene of a subject comprising:
providing a DNA sample from said subject;
contacting said DNA sample with at least four primer sets;
generating an extension product from each of said at least four primer sets comprising a region of DNA that includes the location of a genetic marker;
grouping said extension products according to the following grouping assignments, wherein at least one group includes at least four pooled extension products:
Group 1 comprising extension products generated from primer sets SEQ ID NOs 358-359, SEQ ID NOs 364-365, and SEQ ID NOs 186-187;
Group 2 comprising extension products generated from primer sets SEQ ID NOs 235-236, SEQ ID NOs 305-306, SEQ ID NOs 226-227;
Group 3 comprising extension products generated from primer sets SEQ ID NOs 262-263, SEQ ID NOs 330-331, SEQ ID NOs 319-320, SEQ ID NOs 221-222, and SEQ ID NOs 325-326;
Group 4 comprising extension products generated from primer sets SEQ ID NOs 267-268, SEQ ID NOs 203-204, SEQ ID NOs 230-231, SEQ ID NOs 191-192, SEQ ID NOs 284-285, SEQ ID NOs 219-220, and SEQ ID NOs 205-206;
Group 5 comprising extension products generated from primer sets SEQ ID NOs 376-377, SEQ ID NOs 378-379, SEQ ID NOs 280-281, SEQ ID NOs 237-238, SEQ ID NOs 335-336, SEQ ID NOs 312-313, SEQ ID NOs 255-256, SEQ ID NOs 330-331, SEQ ID NOs 277-278, SEQ ID NOs 235-236, SEQ ID NOs 189-190, SEQ ID NOs 282-283, and SEQ ID NOs 248-249;
Group 6 comprising extension products generated from primer sets SEQ ID NOs 269-270 SEQ ID NOs 257-258, SEQ ID NOs 198-199, and SEQ ID NOs 337-338;
Group 7 comprising extension products generated from primer sets SEQ ID NOs 271-272, SEQ ID NOs 328-329, SEQ ID NOs 314-315, SEQ ID NOs 296-297, SEQ ID NOs 242-243, SEQ ID NOs 367-368, SEQ ID NOs 182-183, and SEQ ID NOs 360-361;
Group 8 comprising extension products generated from primer sets SEQ ID NOs 371-372, SEQ ID NOs 341-342, SEQ ID NOs 355-356, SEQ ID NOs 339-340, and SEQ ID NOs 298-299;
Group 9 comprising extension products generated from primer sets SEQ ID NOs 273-274 and SEQ ID NOs 323-324;
Group 10 comprising extension products generated from primer sets SEQ ID NOs 369-370, SEQ ID NOs 321-322, SEQ ID NOs 228-229, and SEQ ID NOs 212-213;
Group 11 comprising extension products generated from primer sets SEQ ID NOs 210-211, SEQ ID NOs 383-384, and SEQ ID NOs 381-382;
Group 12 comprising extension products generated from primer sets SEQ ID NOs 275-276;
Group 13 comprising extension products generated from primer sets SEQ ID NOs 175-176 and SEQ ID NOs 177-178;
separating said extension products on the basis of melting behavior; and
identifying the presence or absence of said genetic markers in said subject by analyzing the melting behavior of said extension products.
2. The method of claim 1 , wherein said at least four primer sets comprise at least five primer sets.
3. The method of claim 2 , wherein said at least five primer sets comprise at least six primer sets.
4. The method of claim 3 , wherein said at least six primer sets comprise at least seven primer sets.
5. The method of claim 4 , wherein said at least seven primer sets comprise at least eight primer sets.
6. The method of claim 1 , wherein the extension products generated from said primer sets are grouped with those having similar melting behaviors.
7. The method of claim 1 , wherein extension products within groups are separated on a same lane of a TTGE gel or in the same run on a DHPLC.
8. The method of claim 2 , wherein the extension products generated from said primer sets are grouped with those having similar melting behaviors and separated on the basis of melting behavior on a same lane of a TTGE gel or in the same run on a DHPLC.
9. The method of claim 3 , wherein the extension products generated from said primer sets are grouped with those having similar melting behaviors and separated on the basis of melting behavior on a same lane of a TTGE gel or in the same run on a DHPLC.
10. The method of claim 4 , wherein the extension products generated from said primer sets are grouped with those having similar melting behaviors and separated on the basis of melting behavior on a same lane of a TTGE gel or in the same run on a DHPLC.
11. The method of claim 5 , wherein the extension products generated from said primer sets are grouped with those having similar melting behaviors and separated on the basis of melting behavior on a same lane of a TTGE gel or in the same run on a DHPLC.