Se33 mutations impacting genotype concordance

Abstract

Disclosed are primer set compositions, methods and kits for human identification using the highly complex sequence locus, SE33 (ACTBP2) in single and multiplex PCR reactions. Additionally, disclosed are three newly discovered single nucleotide polymorphisms (SNPs) within the SE33 locus that can cause discordance seen as mobility shift or allelic dropout. Also disclosed are kits useful in human identification.

1 . A nucleic acid composition comprising a single nucleotide polymorphism (SNP), wherein said SNP is located at one of positions 316, 317 or 324 as illustrated in SEQ ID NO:3, and wherein said SNP is 3′ of the short tandem repeat (STR) region of the SE33 locus. 
     
     
         2 . The nucleic acid composition of  claim 1 , wherein the SNP at position 316; wild type nucleotide is C and variant nucleotide is T, at position 317; wild type nucleotide is G and variant nucleotide is A and at position 324; wild type nucleotide is G and the variant nucleotide is A. 
     
     
         3 . The nucleic acid composition of  claim 2 , wherein when an amplification product contains one of the variant SNP a mobility shift is seen by capillary electrophoresis. 
     
     
         4 . The nucleic acid composition of  claim 3 , wherein the mobility shift in the amplification product results from a destabilized hairpin between nucleotides 314 and 326. 
     
     
         5 .- 13 . (canceled) 
     
     
         14 . An amplification primer set for SE33 locus comprising:
 a. a first primer capable of annealing to a primer-binding site 5′ of SE33's STR region; and   b. a second primer, a reverse primer, capable of annealing to a first primer-binding site 3′ of the SE33 STR region, wherein the first primer-binding site comprises one variant SNP, wherein the variant SNP is selected from one of nucleotide positions 316, 317 and 324 as illustrated in SEQ ID NO:3.   
     
     
         15 . The amplification primer set of  claim 14 , further comprising at least a third primer, a second reverse primer, capable of annealing to a second primer-binding site 3′ of the SE33 STR region. 
     
     
         16 . The amplification primer set of  claim 15 , wherein the first reverse primer binding site and the second reverse primer binding sites 3′ of the SE33 STR region share at least 10% sequence identity. 
     
     
         17 . The amplification primer set of  claim 15 , wherein the first reverse primer binding site and the second reverse primer binding site 3′ of the SE33 STR region are different. 
     
     
         18 . The amplification primer set of  claim 15 , wherein the 3′ terminus of the third primer extends at least 1 to at least 30 nucleotides towards the 5′ end of a template from the variant SNP position. 
     
     
         19 . The amplification primer set of  claim 15 , wherein at least one reverse primer can further comprise at least one nucleotide analog or at least one universal base or at least one modified nucleotide or at least one deaza-G nucleotide or can be a degenerate reverse primer. 
     
     
         20 . A kit for analyzing a plurality of STR loci in a nucleic acid sample, wherein the kit comprises at least a first SE33 STR locus primer set, wherein the SE33 primer set generates a first amplification product for said nucleic acid sample comprising a SNP at one of positions 316, 317 or 324 as illustrated in SEQ ID NO:3. 
     
     
         21 . The kit of  claim 20 , wherein the SNP at one of positions 316, 317 or 324 is a variant SNP. 
     
     
         22 . The kit of  claim 20 , wherein the SE33 STR locus primer set further comprises a third primer, a second reverse primer for the SE33 locus. 
     
     
         23 . The kit of  claim 22 , wherein the amplification product for the SE33 primer set comprises a second amplification product. 
     
     
         24 . The kit of  claim 23 , wherein the second amplification product lacks a SNP at one of positions 316, 317 or 324. 
     
     
         25 . The kit of  claim 23 , wherein the second amplification product includes a SNP at one of positions 316, 317, or 324. 
     
     
         26 . The kit of  claim 25 , wherein the SNP is a variant SNP. 
     
     
         27 . The kit of  claim 20 , further comprising at least one SE33 control DNA, wherein said control DNA comprises one variant SNP located at one of positions 316, 317 or 324 as illustrated in SEQ ID NO:3. 
     
     
         28 . The kit of  claim 20 , further comprising at least one additional STR locus primer set selected from vWA, CSF1PO, TPOX, D5S818, D7S820, D13S317, D16S539, D8S1179, D21S11, D18S51, TH01, FGA, D3S1358, and one or more additional STR locus primer set selected from D19S433, D2S1338, D10S1248, D22S1045, D2S441, D1S1656, CSF, D6S1043, D10S1248, Penta D, Penta E, D3S1744, D7S1517, D10S2325, D21S2055, DYS391, and D12S391. 
     
     
         29 . The kit of  claim 20 , further comprising a primer set for Amelogenin. 
     
     
         30 . The kit of  claim 20 , wherein a second SE33 STR locus primer set is present in the kit or is present in a different kit. 
     
     
         31 . The kit of  claim 28 , wherein the STR locus primer sets are amplified in a multiplex amplification reaction.