Determination of ACTN3 genotype

Background

This protocols is adopted from Schadock et al. (2015)1 and is suitable for determining the ACTN3 genotype.

Materials

  • A PCR machine
  • Specific primers

Primers used un Schadock et al. 

Primer sequence Final reaction concentrations Volume of 100 uM in 500 ul primer mix
hsACTN3_F1 CGCCCTTCAACAACTGGCTGGA 0.5 uM 10 ul
hsACTN3_R1 GATGAGCCCGAGACAGGCAAGG 0.5 uM 10 ul
hsACTN3Tif_F2 CAACACTGCCCGAGGCTGACTG 0.125 uM 2.5 ul
hsACTN3Cir_R2 CATGATGGCACCTCGCTCTCGG 0.25 uM 5 ul

Add primers to 472.5 ul nuclease free H2O to make a 4X primer mix.

  • PCR master mix
  • PCR reactions tubes

Methods

  • Extract DNA (from whole blood or muscle)
  • Mix sample, master mix and primer mix per reaction in a PCR reaction tube:
Component Volume
2X Master mix 10 ul
Primer mix 5 ul
Sample (correspondning to 100-250 ng genomic DNA) 5 ul
  • Run the PCR reaction
Time Temperature Cycles
2 min 95°C
10 sec 95°C
10 sec 68°C 35 cycles
45 sec 72°C
2 min 72°C
  • Run the PCR product on a 2% agarose gel using 2-5 ul of the reaction mix. Use a DNA ladder and H2O as negative control.

Analysis

A R/R genotype produces two bands at 690 and 413 bp. A X/X genotype produces two bands at 690 and 318 bp.

Footnotes

  1. Schadock, I., et al. (2015). “Simple Method to Genotype the ACTN3 r577x Polymorphism.” Genet Test Mol Biomarkers 19(5): 253-257.↩︎