Mice Genotyping Using Buccal Swab Samples: An Improved Method

Abstract

Routine methods used to genotype mice involve isolation of DNA from partially amputated neonate's tail, toe or ear. Inevitable drawback of such techniques is animal`s pain response and increased spending of time and funds because of obligatory DNA purification. In order to implement a noninvasive and simple protocol for mouse DNA isolation, we have improved the method based on samples collected by swabbing of the inner cheek. Combining alkaline and temperature lysis it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method on three different mouse lines showed that it is highly efficient, the volume of the PCR samples could be reduced to 25 µl and fragments up to 800 bp were successfully amplified. This protocol reduces animal discomfort, shortens the time for DNA isolation, and enables amplification of larger DNA fragments with optimal success rate, thus considerably facilitating large-scale genotyping of different mouse lines.Key words: DNA isolation, buccal swabs, genotyping, PCR, mouse Transgenic mice are an indispensable experimental model in biomedical research and their successful breeding requires a simple and reliable genotyping procedure, which is typically performed using PCR (polymerase chain reaction). Obtaining high quality genomic DNA is a critical for successful PCR amplification. Although large quantities of DNA can be isolated from a variety of samples, the routinely used methods for mouse genotyping involve partial amputation of a neonate's tail, ear or toe (Hanley et al. 1991, Ren et al. 2001, Malumbres et al. 1997. Although efficient, the procedures are invasive, mutilating and the animals regularly exhibit pain response. Moreover, additional step of DNA purification after tissue lysis represents a significant burden, especially in laboratories which need to genotype a large number of different samples. Therefore, every simplification of DNA isolation procedure is welcomed, as it follows the trend in refinement of animal handling procedures, and also saves both time and money (Council of Europe, 2006.).Buccal swabs as a source of cells for DNA isolation was used to avoid the invasiveness of previously applied procedures. After swabbing of buccal mucosa, DNA could be isolated using commercially Here we report an improved simple method (Meldgaard et al. 2004) for mouse genotyping using buccal swab samples. By modifying the alkaline lysis and boiling method it was possible to reliably isolate the DNA from mouse buccal mucosa using simple cotton sticks and the basic, easily affordable chemicals. The PCR was performed in minimal volume (25 µL) and modified in order to achieve amplification of PCR products up to 800 bp. MATERIALS AND METHODS Animals and housing Sampling of buccal epithelial cells and isolation of DNAThe sampling of the buccal mucosa required some skilfulness and routine, as the sample should contain cells of buccal mucosa and not those of tongue. The mice were firmly held in the hand with their neck skin fixed between the thumb and the...

DNA from tissues of young mice is optimal for genotyping

“…However, non-invasive methods have also been described recently [11,12,13,14,15]. Moreover, inadequate amounts and/or poor quality of DNA extracted for genotyping can be a problem if large amounts are required and if animals have to be subjected to repeat analyses.…”

“…Moreover, we obtained only relatively small amounts of DNA in terms of both volume and concentration. There are, however, reports [11,12] of the reliable and efficient isolation of DNA from buccal swabs by other methods that yield free from inhibitors of PCR.…”

“…Several methods of non-invasive genotyping have been proposed, such as sampling of saliva (by oral swabs) [11,12], hair [13] and fecal pellets [14,15], and methods for extraction of DNA from such animal samples are constantly improving [16].…”

PCR-Quality DNA Isolation from Human Bronchial Aspirates and Buccal and Eyelid Swabs by a Simple Procedure Based on Alkaline Lysis

“…The initial starting point for DNA isolation was the procedure used for mouse buccal swabs that combined alkaline lysis and boiling (Mitrečić et al, 2008). As the initial testing of buccal swabs with this procedure was successful, simplification of this technique was attempted.…”

“…Alkaline lysis as a simple method for DNA isolation was already used in the analysis of samples from human and mouse buccal swabs (Rudbeck and Dissing, 1998;Meldegaard et al, 2004;Mitrečić et al, 2008). It is based on solubilization of membrane proteins, which enables the unaffected DNA to leave the cells and be available for further analysis.…”