DNA extraction is a routine procedure of molecular biology used in research laboratories and forensic analyses. The process is composed of a combination of physical and chemical steps [1]. Solution-based DNA extraction methods using organic solvents consists of the organic extraction and subsequent salting-out. The chemical steps include lysis, phenol-chloroform extraction, ethanol precipitation and washing. Organic extraction is mostly used in laboratories because it is affordable and allows the production of large quantities of pure DNA. The salting out method exploits the dependence of protein solubility on their chemical-physical characteristics, temperature, pH and saline concentration of the solution. Low salt concentrations increase protein solubility (salting-in), while high salt concentrations (e.g.: NaCl 3M) decrease it (salting-out) leading to protein precipitation. These two methods involve the use of toxic chemicals, like phenol and chloroform, and increase the risk of contamination because of the DNA transfer between multiple tubes. Other procedures of DNA isolation, such as commercially available kits (e.g. "QIAamp DNA Mini Kit" from Qiagen, Germany or "PureLink Genomic DNA Mini Kit" from Invitrogen, USA), Chelex 100 extraction (a chelating resin with a high affinity for polyvalent metal ions such as magnesium) and Solid Phase Extraction (SPE) are based on chromatographic principles. These methods differ in quality and quantity of extracted DNA but are all very expensive and poorly reproducible.
The choice of the most suited DNA extraction method depends on several factors, including cost, time, easiness of execution, quality and quantity of DNA isolated, safety and risk of contamination. In addition, the type of sample from which the DNA is extracted must also be considered [2, 3]. DNA can be extracted from diverse sources of samples through different methods that depend on the selected tissue. In animals, peripheral blood leukocytes are the usual source of genomic DNA [4]. The most common blood collection procedures in mice (e.g. withdrawal from the orbital sinus) are usually painful and stressful for the animal; the performing personnel must be appropriately trained and compliant with guidelines for anesthesia and/or analgesic use and maximal sample volumes and phlebotomy frequency [5].
In detail, blood collection volume should not exceed 10% of total blood volume. Common DNA extraction methods require large amounts of blood (about 500 µL), but mice have on average about 55 mL of blood per kg of bodyweight, so usually a mouse only has few milliliters of blood in total. For this reason, it is difficult to get satisfactory results with commercial kits.
Different mouse models are commonly used for the study of diabetes, including the non-obese diabetic (NOD) mice, a useful animal model for the study of the immunopathogenetic events leading to autoimmune type 1 diabetes development, and the obese mice (Lepob, referred to as ob or ob/ob) and their genotyping is of paramount importance. In the “single nucleotide polymorphism-restriction fragment length polymorphism” (SNP-RFLP) enzymatic method a region containing a specific SNP allele is targeted and amplified by PCR and subsequently digested using an endonuclease enzyme [6]; the length variation of the digested PCR fragments is then distinguished by gel electrophoresis [7]. It is a relatively simple, rapid, inexpensive, and convenient method frequently used for to the detection of those small genetic differences between individuals which can lead to physiological or pathological changes [8–9].
In NOD/LtJ mice, a spontaneously G→T transversion mutation (designated as Leprdb−5J) occurs at position 640 of the leptin receptor gene (at the protein level, the mutation causes a valine-to-glycine substitution) and mutant mice become obese and hyper-insulinemic [10, 11].
We have here developed an up-dated method for the extraction of genomic DNA from mouse whole blood, obtained by puncturing the tail tip, and letting blood drops to be adsorbed and dry on Whatman 3 MM paper. This method may substantially facilitate SNP-based genotyping by presenting many advantages such as: cost reduction, the employment of a less invasive practice for blood sample collection in small laboratory animals and the necessity of reduced blood volumes (about 50 µL); furthermore, it also allows easy blood transportation among laboratories. We have tested this method for NOD Leprdb5j mice genotyping.