Customized DNA Primers used to amplify Amelogenin and 4 STR loci inspired from CODIS using standard and multiplex PCR.
Although the PAGE version of EasyTaq would have been available to us, our lab isn’t equipped for polyacrylamide gel electrophoresis (PAGE). Therefore, I chose 4 STR markers plus X (male) and Y (female) markers and redesigned the DNA oligonucleotide sequences in a manner that I could maximize the difference in PCR product sizes for each STR and analyze them using standard 3$ agarose gel electrophoresis. Despite this, it remains unlikely that small variations in single tandem repeats (i.e 3-9 bp) will be observable on an agarose gel.
Primer sequences are either identical or inspired from the following CODIS STR loci :
- D14S608 (426bp)
- D15S659 (284bp)
- D7S3048 (234 bp)
- D18S535 (a: 175bp; b: 201bp)
For AMEL X and Y loci, the DNA primers were not designed so that they would discriminate between chromosome X and Y based on PCR amplicon size. Instead, the oligonucleotides were designed to specically amplify either AMELX or AMELY based on their respective genomic DNA sequences.
F1.1 AMELX : CCATTGTTTGCGTTAACAATGC (127 bp amplicon)
F1.2 AMELY : ATCACTGTTTGCATTAGCAGTCC (134 bp amplicon)
R1.1xy_AMEL : ATCAGAGCTTAAACTGGGAAGCTG
* Nucleotides in bold represent mismatches between X and Y sequences in the Amelogenin gene.
All corresponding DNA files can be downloaded in Snapgene format here.