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The journey of my dog’s sample in the genetic lab. Step 2

10.12.2020

Step two: Genetic test   A genetic test is an analysis of animal’s DNA used to investigate changes that cause or increase the likelihood of developing specific diseases, responses to therapy, physical characteristics, and the identity of the animal. DNA is a genetic record or code with instructions for functioning of an organism. When a biological sample of your animal (most commonly blood or swab) arrives in the laboratory the first step is DNA isolation. We have already written more about the process of DNA isolation previously (available here). In this article we will describe the process of the genetic test itself.     We use various laboratory techniques and high-throughput laboratory equipment to perform genetic tests. To be able to analyse changes in a DNA molecule, it is necessary to multiply the regions of interest to the limit of detection. Basic method for DNA amplification and genetic testing is the PCR method - polymerase chain reaction. The PCR method is used to amplify specific segments on DNA to enable the detection or measurement of changes in genes or regions. The DNA sample of your animal is used as a template for multiplication.   Analysis of the amplified DNA segments can be performed using various laboratory techniques selected according to the type of DNA change being investigated. The most used methods are DNA sequencing (determination of the nucleotide sequence of DNA), genotyping using real-time PCR and DNA analysis by gel electrophoresis. In all techniques, amplified segments of DNA are stained with different dyes to enable the detection of genetic changes by laboratory instruments.   In our laboratory, we use reference methods to examine or test one gene at a time. In most cases, a genetic test is used to analyse one change that is related to a specific genetic disease or trait. In some diseases or traits, multiple changes in the same gene or changes in multiple genes may be involved. In this case, it is necessary to perform several individual tests to analyse all changes that were related to the investigated genetic disease or trait and get the final result of the genetic test. An example of a genetic test that consists of several individual tests is the brown coat colour test, which includes 3 or in some breeds even 4 individual tests.   When performing genetic tests quality control is routinely performed to control the performance of individual steps of a genetic test. Appropriate control samples and negative controls are used to control the performance of a genetic tests and ensure that the result of the analysis is correct. In addition to daily quality control, international interlaboratory controls are also organized and enable laboratories to compare their results with the results of other laboratories and obtain a certificate for their participation (ISAG). Laboratories can also prove an appropriate quality management system with an ISO certificate, which they obtain based on an annual inspection by the relevant certification body.

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The journey of my dog’s sample in the genetic lab

13.11.2020

Step one: DNA isolation   All living creatures have genetic material in their cells that determines their characteristics and distinguishes them from each other. In animals, the carrier of genetic information is DNA (deoxyribonucleic acid). DNA molecule consists of a sequence of several billions of subunits called nucleotides, which, similar to the sequence of letters in books, gives instructions for the functioning of the organism. The amount of data in DNA within the subject is enormous and it cannot be routinely screened entirely. Therefore we use DNA tests to analyse DNA segments of interest.   An essential step in DNA testing is DNA isolation. It is the technique used to extract DNA molecule from a biological sample. The quantity, quality and integrity of DNA depend on the quality of the biological sample. It is crucial that the sample is as fresh as possible and contains a sufficient amount of biological material. Sample quality assessment is performed upon the admission to the laboratory and before DNA isolation. In our laboratory, DNA is mostly extracted from blood and buccal swab of an animal. A few drops of blood or a swab of the oral mucosa without visible mould and/or food debris are usually enough to successfully isolate DNA.     In animal cells, DNA molecule is organized in chromosomes within the cell nucleus. DNA isolation therefore involves physical and chemical approaches that allow DNA to be released from the cells and to be separated from other cellular components. DNA isolation process takes about two hours, can be manual or automated, and consists of three basic steps: lysis, precipitation, and purification. Lysis involves the mechanical disruption of cells and the enzymatic dissolution of cellular proteins. In the precipitation process, the alcohol is added to separate the released DNA from the cellular debris. In the last step, the released DNA is purified by washing away cell debris and reagents and stored at -20°C. Properly isolated and stored DNA can be used for genetic testing for many years.   A good quality DNA is crucial for successful performance of a genetic test. To be able to isolate good quality DNA in the laboratory, we need appropriate biological sample, therefore it is important to strictly follow the sample collection instructions available here. If DNA of sufficient quality cannot be isolated from the sample, a new sample is required to perform the genetic test.