DNA genotyping: Creating trust
Human beings are curious by nature and it is this curiosity that leads to new discoveries in all spheres of society. One of these discoveries that resulted in the use of inherited genetics as the basis of individual identification of different organisms by using various techniques and methodologies, is DNA genotyping. The “unique” genetic fingerprint generated using DNA genotyping can be used to confirm identity and perform parentage analysis of an organism given that the methodology used will allow sufficient discrimination between individuals in the population of interest. To achieve this, the uniqueness of the DNA fingerprint needs to be such that the probability of randomly obtaining the same fingerprint in the population of interest should be close to zero. DNA fingerprinting using Short Tandem Repeats (STR; also known as microsatellite analysis) allows for a very high degree of individual discrimination and is currently the gold standard for forensic identification purposes for humans and a wide variety of other mammals. This technique relies on a number of specific marker regions to be targeted in the genome and the target regions are chosen based on the polymorphic properties observed in the species or population of interest. Figure 1 represents an example of 3 marker regions amplified from DNA inherited from each parent to generate a DNA fingerprint represented by the sizes obtained for each marker region. It is these sizes that are used to generate a DNA profile (as listed in the DNA profile certificate) used for parentage analysis.
Figure 1. Graphical representation of 3 marker regions targeted for STR DNA amplification and analysis. The blue and pink lines represent DNA regions inherited from the father and the mother, respectively. The black, green and yellow block inserts in the paternal and maternal DNA regions represents 3 different STR regions. The 3 markers will yield a homozygous genotype for the black STR repeats (eg. 164; 164), a heterozygous genotype for the green STR repeats (eg. 162; 170) and a heterozygous genotype for the yellow STR repeat (eg. 166; 168). Using the genotypes obtained from the offspring, allows for identification of the parent(s) from which the offspring inherited the DNA simply by comparing their DNA profiles for the same marker regions.
STR analysis of the sample
STR analysis in cattle is routinely used for identification and parentage analysis throughout the world. Minute quantities starting materials are required due to the fact that the process is based on polymerase chain reaction (PCR) for the highly efficient amplification of the STR marker regions. The International Society for Animal Genetics (ISAG) routinely performs evaluations of different marker regions to establish a minimum recommended marker panel to be used for routine identification and parentage testing. ISAG recommended 9 STR markers in 2006 and 12 STR markers since 2009 to improve the accuracy of parentage and increase the confidence in the results obtained. Some laboratories now make use of the standard 12 STR markers, but include additional polymorphic STR markers (based on literature) to further improve the confidence in the results in order to establish trust in the DNA genotyping service industry. The impact of the additional number of highly polymorphic markers becomes clear when only a single parent (usually sires from a multi-sire breeding programme) needs to be identified as the qualifying parent of the offspring in question. The chance of random inclusion of a single parent (especially genetically related sires in the absence of a dam) as a qualifying parent to a tested offspring is significantly higher than when a parent pair is used for parentage analysis, often resulting in the testing laboratory requesting a sample from the dam in order to reduce the number of qualifying sires. The additional testing is usually associated with additional cost in terms of time and money and causes unfair distrust towards a solid and proven technology. Laboratories can alleviate this problem through the use of an extended marker panel (12 ISAG and additional markers) for routine parentage at no additional cost to the client. Personal experience has shown that a 17 marker panel (12 ISAG and 5 FAO markers), when compared to the standard 12 marker ISAG panel, used for routine identification and parentage, resulted in a significant increase in the ability to identify single qualifying sires from a multi-sire breeding programme when tested against potential offspring.
Next generation technologies
The advancement of DNA analysis technologies during the past few years has been unsurpassed and the development and analysis costs associated with these technologies have been out-pacing Moore’s law (used as a measure of progress). The successful use of these next generation technologies and current technologies are based on good quality input DNA. It is therefore imperative that high quality, high molecular weight DNA gets isolated from supplied samples to yield “future” ready DNA and that the integrity of the DNA is maintained for future use. This is only possible if the necessary care is taken when sampling is performed and that the sample is handled appropriately to prevent DNA degradation prior to receipt by the DNA genotyping laboratory. To ensure that your sampling procedure and samples meets the necessary requirements for next generation DNA analysis, it is advisable to consult with a DNA genotyping service laboratory.
Trust in DNA genotyping
Trust in the use of DNA technology can only be earned and sustained in a partnership between the client and the service laboratory. As a laboratory it is imperative that all steps are taken to ensure that results are reproducible and there is full traceability of the sample from receipt to result. Reproducibility is achieved through adhering to internationally accepted quality standards and traceability by a robust sample management process that records, in detail, the progress and verification steps of any sample from receipt to result. This process can also be improved through automation where humans were traditionally involved. This, from a laboratory’s perspective, is how reproducibility and traceability is “ensured” and that the sample received is the one actually reported on. It must however be emphasized that accurate results received from a DNA genotyping service laboratory is dependent on them receiving correctly identified samples and associated information. By adhering to the before mentioned best practices, a DNA genotyping service laboratory will be able to efficiently and accurately verify any breeding programme, thereby allowing the breeder to sell DNA-verified pedigree animals with confidence and earning the trust from the breeding community.
We at Clinomics believe in providing answers to client questions through the use of DNA information obtained from proven technologies, while keeping our clients’ samples DNA ready for next generation DNA analysis.
Michel Labuschagné (PhD)