SE33 mutations impacting genotype concordance
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) at least one forward primer capable of annealing to SE33 locus 5′ of a STR region; and
b) at least a first reverse primer capable of annealing to the SE33 locus 3′ of the STR region, wherein the at least a first reverse primer comprises at least 20 contiguous nucleotides comprising positions 316, 317, and 324 of the inverse complement of SEQ ID NO:3, wherein SEQ ID NO: 3 comprises an A at position 317 or an A at position 324 and wherein the at least one forward primer comprises a fluorescent label.
2. A kit for analyzing a plurality of STR loci in a nucleic acid sample, the kit comprising at least a first SE33 STR locus primer set capable of generating a first amplification product, wherein:
a first primer is capable of annealing to SE33 locus 5′ of a STR region,
a second primer comprises at least 20 nucleotides comprising positions 316, 317, and 324 of the inverse complement of SEQ ID NO:3,
SEQ ID NO: 3 comprises an A at position 317 or an A at position 324, and
at least one of the first or second primer comprises a fluorescent label.
3. The kit of claim 2 , wherein the SE33 STR locus primer set further comprises a third primer, a second reverse primer for the SE33 locus.
4. The kit of claim 2 , 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 of SEQ ID NO:3.
5. The kit of claim 2 , further comprising at least one additional STR locus primer set selected from vWA, CSF1PO, TPDX, 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.
6. The kit of claim 2 , further comprising a primer set for Amelogenin.
7. The nucleic acid composition of claim 1 , wherein SEQ ID NO:3 further comprises a T at position 316.
8. The nucleic acid composition of claim 1 , further comprising a second reverse primer for the SE33 locus.
9. The nucleic acid composition of claim 8 , wherein the first reverse primer, the second reverse primer, or both comprise at least one nucleotide analog.
10. The nucleic acid composition of claim 8 , wherein the first reverse primer, the second reverse primer, or both comprise at least one universal base.
11. The nucleic acid composition of claim 8 , wherein the first reverse primer, the second reverse primer, or both comprise at least one modified nucleotide.
12. The nucleic acid composition of claim 8 , wherein the first reverse primer, the second reverse primer, or both comprise at least one deaza-G nucleotide.
13. The nucleic acid composition of claim 8 , wherein the first reverse primer, the second reverse primer, or both comprise are degenerate.
14. The kit of claim 2 , wherein SEQ ID NO:3 further comprises a T at position 316.
15. The kit of claim 3 , wherein the second primer, the third primer, or both comprise at least one nucleotide analog.
16. The kit of claim 3 , wherein the second primer, the third primer, or both comprise at least one universal base.
17. The kit of claim 3 , wherein the second primer, the third primer, or both comprise at least one modified nucleotide.
18. The kit of claim 3 , wherein the second primer, the third primer, or both comprise at least one deaza-G nucleotide.
19. The kit of claim 3 , wherein the second primer, the third primer, or both are degenerate.