Differential amplification of alleles: Potential for misclassification with PCR genotyping

Abstract

Abstract Growth hormone genotype was determined using polymerase chain reaction (PCR) amplification of the growth hormone gene. Amplified DNA was digested with MspI, the resulting fragments separated by agarose gel electrophoresis and visualized with UV light following ethidium bromide staining. Undesired preferential amplification of alleles initially led to genotype misclassification. Two of 32 animals were misclassified when PCR products were compared to genomic digests probed with radiolabeled, full‐length cDNA of the bovine growth hormone gene. Intensity of allelic bands was quantified by densitometry. One allelic form (A1) consistently exhibited greater band intensity. Relative intensity of the A1 allele to the A2 allele varied from 1.5:1 to 18:1, depending on size of PCR amplified DNA and length of the 72°C phase of PCR. Differential intensity increased with increasing size of PCR amplified DNA and length of the 72°C phase of PCR. Subsequent analyses indicated that the apparent differential amplifi...