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 method comprising:
a. providing a forward primer capable of annealing to SE33 locus 5′ of a short tandem repeat region,
b. providing a reverse primer, the reverse primer comprising 10 or more contiguous nucleotides of the inverse complement of positions 316, 317, and 324 of SEQ ID NO: 3, wherein SEQ ID NO 3 comprises a T at position 316, A at position 317, or A at position 324,
c. amplifying a nucleic acid with the forward and reverse primers,
d. separating the amplification products by capillary electrophoresis,
e. comparing the amplification product to an allelic ladder, and
f. identifying the presence of the amplification products.
2. The method of claim 1 , wherein at least one primer comprises a label.
3. The method of claim 1 , wherein said amplifying comprises using a polymerase chain reaction (PCR) to generate an amplification product.
4. The method of claim 1 , further comprising a second reverse primer.
5. The method of claim 1 , further comprising primers for the loci D10S1248, vWA, D16S539, D2S1338, amelogenin, D8S1179, D21S11, D18S51, D22S1045, D19S433, TH01, FGA, D2S441, D3S1358, D1S1656 and D12S391.
6. The method of claim 1 , thither comprising a size standard.