Multiplex amplification of short tandem repeat loci

Abstract

Methods and materials are disclosed for use in simultaneously amplifying at least thirteen loci of genomic DNA in a single multiplex reaction, as are methods and materials for use in the analysis of the products of such reactions. Included in the present invention are materials and methods for the simultaneous amplification of at least thirteen short tandem repeat loci, including specific materials and methods for the analysis of thirteen such loci specifically selected by the United States Federal Bureau of Investigation as core loci for use in the Combined DNA Index System (CODIS) database.

1 . A method of simultaneously determining the alleles present in a set of loci from one or more DNA samples, comprising: 
 (a) obtaining at least one DNA sample to be analyzed,    (b) selecting a set of loci of the DNA sample, comprising at least thirteen short tandem repeat loci which can be co-amplified,    (c) co-amplifying the loci in the set in a multiplex amplification reaction, wherein the product of the reaction is a mixture of amplified alleles from each of the co-amplified loci in the set; and    (d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample.    
     
     
         2 . (canceled)  
     
     
         3 . (canceled)  
     
     
         4 . The method of  claim 1 , wherein at least one of the at least thirteen short tandem repeat loci in the set of loci selected in step (b) is a pentanucleotide tandem repeat locus.  
     
     
         5 . The method of  claim 4 , wherein the pentanucleotide tandem repeat locus is selected from the group consisting of G475, C221, and S159.  
     
     
         6 . The method of  claim 1 , wherein the set of loci selected in step (b) further comprises a locus which can be used to identify the gender of at least one source of the DNA provided in step (a).  
     
     
         7 . The method of  claim 6 , wherein the at least one source of the DNA provided in step (a) is a human being, and the locus used to identify the gender of the human being is an Amelogenin locus.  
     
     
         8 . The method of  claim 7 , wherein the Amelogenin locus is co-amplified using an oligonucleotide primer having a sequence selected from the group consisting of SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:105.  
     
     
         9 . The method of  claim 1 , wherein the multiplex amplification reaction is done using at least one oligonucleotide primer having a sequence selected from at least one of the groups of sequences consisting of: 
 SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80 and SEQ ID NO:81, when one of the loci in the set is D7S820;    SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82, and SEQ ID NO:83, when one of the loci in the set is D13S317;    SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, when one of the loci in the set is D5S818;    SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:49, when one of the loci in the set is D3S1539;    SEQ ID NO:9, SEQ ID NO:10, when one of the loci in the set is D17S1298;    SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:52, SEQ ID NO: 53, when one of the loci in the set is D20S481;    SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 55, SEQ ID NO: 61, when one of the loci in the set is D9S930;    SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:54, when one of the loci in the set is D10S1239;    SEQ ID NO:17, SEQ ID NO:18, when one of the loci in the set is D14S118;    SEQ ID NO:19, SEQ ID NO:20, when one of the loci in the set is D14S562;    SEQ ID NO:21, SEQ ID NO:22, when one of the loci in the set is D14S548;    SEQ ID NO:23, SEQ ID NO:24, when one of the loci in the set is D16S490;    SEQ ID NO:25, SEQ ID NO:26, when one of the loci in the set is D16S753;    SEQ ID NO:27, SEQ ID NO:28, when one of the loci in the set is D17S1299;    SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, when one of the loci in the set is D16S539;    SEQ ID NO:31, SEQ ID NO:32, when one of the loci in the set is D22S683;    SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:98, when one of the loci in the set is HUMCSF1PO;    SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72 and SEQ ID NO:73, when one of the loci in the set is HUMTPOX;    SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, when one of the loci in the set is HUMTH01;    SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76 when one of the loci in the set is HUMvWFA31    SEQ ID NO:41, SEQ ID NO:42, when one of the loci in the set is HUMF13A01;    SEQ ID NO:43, SEQ ID NO:44, when one of the loci in the set is HUMFESFPS;    SEQ ID NO:45, SEQ ID NO:46, when one of the loci in the set is HUMBFXIII;    SEQ ID NO:47, SEQ ID NO:48, when one of the loci in the set is HUMLIPOL;    SEQ ID NO:50, SEQ ID NO:51, when one of the loci in the set is D19S253; and    SEQ ID NO:56, SEQ ID NO:57, when one of the loci in the set is D4S2368.    SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101, and SEQ ID NO:102, when one of the loci in the set is D18S51;    SEQ ID NO:64 and SEQ ID NO:65, when one of the loci in the set is D21S11;    SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, when one of the loci in the set is D3S1538;    SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, when one of the loci in the set is HUMFIBRA;    SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:104, when one of the loci in the set is D8S1179;    SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:105, when one of the loci in the set is Amelogenin;    SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:94, when one of the loci in the set is G475;    SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:96, when one of the loci in the set is S159; and    SEQ ID NO:99 and SEQ ID NO:100, when one of the loci in the set is C221.    
     
     
         10 . The method of  claim 1 , wherein the amplified alleles are separated prior to evaluating in step (d), using a separation means selected from the group consisting of polyacrylamide gel electrophoresis and capillary gel electrophoresis.  
     
     
         11 . The method of  claim 1 , wherein the multiplex amplification reaction is done using at least thirteen pairs of oligonucleotide primers flanking the at least thirteen loci analyzed.  
     
     
         12 . The method of  claim 11 , wherein the set of loci is co-amplified using a polymerase chain reaction  
     
     
         13 . The method of  claim 11 , wherein each of the loci co-amplified in the multiplex reaction in step (b) is co-amplified using a pair of primers which flank the locus, wherein at least one primer of each pair has a fluorescent label covalently attached thereto.  
     
     
         14 . The method of  claim 13 , wherein at least three of the labeled primers have different fluorescent labels covalently attached thereto.  
     
     
         15 . The method of  claim 1  wherein the at least one DNA sample to be analyzed is prepared from human tissue, wherein the human tissue is selected from the group of human tissue consisting of blood, semen, vaginal cells, hair, saliva, urine, amniotic fluid containing placental cells or fetal cells, and mixtures of any of the tissues listed above.  
     
     
         16 . The method of  claim 1 , wherein the amplified alleles are evaluated by comparing the amplified alleles to a size standard, wherein the size standard is selected from the group of size standards consisting of a DNA marker and a locus-specific allelic ladder.  
     
     
         17 . A method of simultaneously identifying the alleles present in a set of loci of from one or more DNA samples, comprising: 
 (a) obtaining at least one DNA sample to be analyzed,    (b) selecting a set of loci of the DNA sample, comprising short tandem repeat loci D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31;    (c) co-amplifying the loci in the set in a multiplex amplification reaction, wherein the product of the reaction is a mixture of amplified alleles from each of the co-amplified loci in the set; and    (d) evaluating the amplified alleles in the mixture to determine the alleles present at each of the loci analyzed in the set within the DNA sample.    
     
     
         18 . The method of  claim 17 , wherein the multiplex amplification reaction is done using at least one primer for at least one locus in the set of at least thirteen loci selected in step (b), wherein the primer has a sequence selected from one of the groups of primer sequences consisting of: 
 SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101, and SEQ ID NO:102, when one of the loci in the set is D18S51;    SEQ ID NO:64 and SEQ ID NO:65, for the locus D21S11;    SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, for the locus HUMTH01;    SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, for the locus D3S1358;    SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, for the locus HUMFIBRA;    SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72, and SEQ ID NO:73, for the locus HUMTPOX;    SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:104, for the locus D8S1179;    SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76, for the locus HUMvWFA31;    SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:98, for the locus HUMCSF1PO;    SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, for the locus D16S539;    SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and SEQ ID NO:81, for the locus D7S820;    SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82, and SEQ ID NO:83, for the locus D13S317; and    SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, for the locus D5S818.    
     
     
         19 . (canceled)  
     
     
         20 . (canceled)  
     
     
         21 . The method of  claim 17 , wherein the multiplex amplification reaction is a polymerase chain reaction.  
     
     
         22 . The method of  claim 17 , wherein the amplified alleles are evaluated by comparing the amplified alleles to a size standard, wherein the size standard is selected from the group of size standards consisting of a DNA marker and a locus-specific allelic ladder.  
     
     
         23 . The method of  claim 17  wherein the at least one DNA sample to be analyzed is prepared from human tissue, wherein the human tissue is selected from the group of human tissue consisting of blood, semen, vaginal cells, hair, saliva, urine, bone, buccal sample, amniotic fluid containing placental cells or fetal cells, and mixtures of any of the tissues listed above.  
     
     
         24 . A method of simultaneously identifying the alleles present in a set of loci of from one or more human genomic DNA samples, comprising: 
 (a) obtaining at least one human genomic DNA sample to be analyzed,    (b) co-amplifying the loci in a set of at least sixteen loci of the human genomic DNA sample in a multiplex amplification reaction, the set of loci comprising D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and S159, wherein the product of the reaction is a mixture of amplified alleles from each of the co-amplified loci in the set; and    (c) evaluating the amplified alleles in the mixture to determine the alleles present at each of the at least sixteen loci of the human genomic DNA sample.    
     
     
         25 . The method of  claim 24 , wherein the multiplex amplification reaction is done using at least one pair of primers flanking each of the loci in the set of sixteen primers selected in step (b).  
     
     
         26 . The method of  claim 24 , wherein the multiplex amplification reaction is done using at least one primer for at least one locus in the set of at least sixteen loci co-amplified in step (b), wherein the primer has a sequence selected from one of the groups of primer sequences consisting of: 
 SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101, and SEQ ID NO:102, when one of the loci in the set is D18S51;    SEQ ID NO:64 and SEQ ID NO:65, for the locus D21S11;    SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, for the locus HUMTH01;    SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, for the locus D3S1358;    SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, for the locus HUMFIBRA;    SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72, and SEQ ID NO:73, for the locus HUMTPOX;    SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:104, for the locus D8S1179;    SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76, for the locus HUMvWFA31;    SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:98, for the locus HUMCSF1PO;    SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, for the locus D16S539;    SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and SEQ ID NO:81, for the locus D7S820;    SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82, and SEQ ID NO:83, for the locus D13S317;    SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, for the locus D5S818;    SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:94, for the locus G475;    SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:96, for the locus S159; and    SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:105, for the Amelogenin locus.    
     
     
         27 . A method of simultaneously identifying the alleles present in a set of loci of from one or more human genomic DNA samples, comprising: 
 (a) obtaining at least one human genomic DNA sample to be analyzed,    (b) co-amplifying the loci in a set of at least sixteen loci of the human genomic DNA sample in a multiplex amplification reaction, the set of loci comprising D3S1358, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO, and C221, wherein the product of the reaction is a mixture of amplified alleles from each of the co-amplified loci in the set; and    (c) evaluating the amplified alleles in the mixture to determine the alleles present at each of the at least sixteen loci of the human genomic DNA sample.    
     
     
         28 . The method of  claim 27 , wherein the multiplex amplification reaction is done using at least one pair of primers flanking each of the loci in the set of sixteen primers selected in step (b).  
     
     
         29 . The method of  claim 27 , wherein the multiplex amplification reaction is done using at least one primer for at least one locus in the set of at least sixteen loci co-amplified in step (b), wherein the primer has a sequence selected from one of the groups of primer sequences consisting of: 
 SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101, and SEQ ID NO:102, for the locus D18S51;    SEQ ID NO:64 and SEQ ID NO:65, for the locus D21S11;    SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:103, for the locus HUMTH01;    SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:106, for the locus D3S1358;    SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:107, for the locus HUMFIBRA;    SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:72, and SEQ ID NO:73, for the locus HUMTPOX;    SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:104, for the locus D8S1179;    SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:76, for the locus HUMvWFA31;    SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:98, for the locus HUMCSF1PO;    SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79, and SEQ ID NO:97, for the locus D16S539;    SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80, and SEQ ID NO:81, for the locus D7S820;    SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82, and SEQ ID NO:83, for the locus D13S317;    SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84, and SEQ ID NO:85, for the locus D5S818;    SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95, and SEQ ID NO:96, for the locus S159;    SEQ ID NO:99 and SEQ ID NO:100, for the locus C221; and    SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:105, for the Amelogenin locus.    
     
     
         30 .- 40 . (canceled)