Common male-specific PCRs
Sry Sex-determining region of the Y chromosome
- forward SRY FÂ : TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA
- reverse SRY RÂ : CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA
- PCR product: 273 base pair
- forward sry1 AACAACTGGGCTTTGCACATTG
- reverse sry2 GTTTATCAGGGTTTCTCTCTAGC
- PCR product: 146/166 bp doublet
Homologous pair SmcX/SmcY
- SMCX-1 5′-CCGCTGCCAAATTCTTTGG-3′
- SMC4-1 5′-TGAAGCTTTTGGCTTTGAG-3′
- PCR product: females single band, males 2 bands because of an intron difference between the X and Y genes (Agulnik 1997, PMID 9060413); adapted from Case Western Transgenic Facility
Test PCR with adult tail genomic samples
Conditions: Templates are known adult male and female tail gDNA samples. Standard PCR cycled using progressively lowered annealing temperature (65-58°C for first cycles, then 58°C); same conditions for all reactions. Primers as listed above. 12μL of a 25μL + 5μL Orange G run on a 2% agarose gel.
Interpretation: The sry F/R pair worked best but does show weak unspecific products of similar size as the male-specific 273bp band. The sry 1/2 pair is probably also specific but bands are harder to recognise. For the SMC, we didn’t observe the described doublet expected in males.
Keep in mind that this is a standard PCR. Amplification and specificity can probably be increased by optimisation of the PCR reaction.