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Amount of Female DNA Contamination Required to Alter a Male DNA Sexing Result Prior to PCR Amplification

Contamination is an important issue that should be addressed when developing any diagnostic procedure. At Avian Biotech a great deal of effort is taken to ensure that every step of the testing process is carefully thought out and performed. Nevertheless, contamination still remains an enormous issue of concern primarily when extremely sensitive assays such as Single-step PCR, Nested Primer PCR, and multiplex PCR are required. Most of our PCR DNA sexing procedures incorporate single-step PCR assays that are sensitive and selective enough to accurately identify the sex from blood or feathers, but allow for the possibility of small amounts of contamination without negatively affecting the final result. Requirements for assays for DNA sexing differ from those developed to identify disease-causing organisms. Identifying infectious disease requires assays which posses the highest levels of sensitivity and selectivity possible. Please refer to exp.2 for more information on different PCR assays for identifying disease causing organisms.

Below are two examples of male DNA samples contaminated with female DNA prior to PCR amplification. Each example indicates the amount of contamination required to alter the final result. All samples were amplified using one of ABI's standard DNA sexing protocol, and run on 1.5 % agarose gel
stained with ethidum bromide.

Fig.1

Fig.1 identifies the minimum amount of female DNA (FDNA) contamination required prior to PCR amplification to alter a male DNA (MDNA) sexing result. In fig.1, levels of FDNA contamination ranged from a 1:256 ratio to a 1:1 ratio. The results of the MDNA samples were not affected below 2ng or a ratio of 8:1 contaminated with FDNA. All contaminated samples contained 16ng of MDNA. Each control sample contained 16ng of either male or female DNA. Fig.1 used primers 2x8 and standard ABI PCR protocol for DNA sexing. Fig.1 indicates that it is virtually impossible, using standard feather sexing protocol, for a female bird to shed enough FDNA onto a male bird to alter a MDNA sexing result.

Note: * Results with an * were not clear and would be considered incomplete and rerun. If a stronger signal was not achieved, a new sample would be requested and run at no extra charge.

Fig.2

Fig. 2 again indicates the amount of female DNA (FDNA) contamination necessary to alter a male DNA (MDNA) sexing result. A false result was not obtained until the contamination ratio was greater than 8:1 or the amount of FDNA present was greater than 2ng. A contamination level of 16:1 or 1ng FDNA did not effect the outcome of the final results. It requires approximately 300 nucleated cells to generate 1ng of genomic DNA. Thus, using standard ABI DNA sexing protocols, it would take more than 1200 female cells to generate the amount of DNA necessary to alter the result of a MDNA sexing sample.

Note: False Result* This result was not clear and would be considered incomplete and rerun. If a stronger signal was not achieved, a new sample would be requested and run at no extra charge.

Both experiments suggested that contamination can play an enormous role in the final outcome of an assay. However, these examples also indicate that the level of contamination required in order to alter a DNA sexing result is relatively high. These and other findings strongly suggest that having male and female specimens in close proximity before and during sample collection will not in any way compromise the accuracy of the final results.

Remember, these finding are based on Avian Biotech's assays. DNA sexing assays from other labs could be more sensitive thus making them more susceptible to contamination errors than ours. Always be sure to use good basic hygiene when collecting samples. The accuracy of each assay begins with proper sample collection techniques.

See results of females sample contaminated with male DNA

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