Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT)

Abstract

Preparation of mouse genomic DNA for PCR is costly and laborious, primarily because of the difficulty in physically separating DNA from other tissue components. This level of purification is unnecessary for many PCR applications. A simple alternative, lysis in an alkaline reagent and neutralization with a suitable buffer, has been used to prepare genomic DNA from buccal swabs, whole blood, semen and forensic samples collected from humans (2). A few modifications of this protocol make it possible to prepare PCR-quality mouse genomic DNA with a brief incubation in hot sodium hydroxide and pH adjustment with a Tris solution (HotSHOT). The HotSHOT method is rapid, inexpensive and may be carried out in 96-well plates, making it amenable to automation and high-throughput genotyping. The reagents for HotSHOT DNA preparation are simple to prepare. An alkaline lysis reagent with 25 mM NaOH, 0.2 mM disodium EDTA and a pH of 12 is prepared by dissolving the salts in water without adjusting the pH. A neutralizing reagent with 40 mM Tris-HCl and a pH of 5 is prepared by dissolving Tris-HCl (not Tris base) in water without adjusting the pH.Tissue samples (neonatal mouse toes, ear punches, 0.2-cm tail snips or 25-mg pieces of spleen) are collected into a 96-well thermal cycler plate or thermal cycler strip tubes. The tissue sample must be small; too large a sample can cause the method to fail. Alkaline lysis reagent (75 μL is added to the samples and heated to 95°C for 10 min to 1 h. The undissolved tissue does not interfere with PCR. After heating, samples are cooled to 4°C, and 75 μL neutralizing reagent are added to each sample. One to five microliters of the final preparation are used per each 10-μL PCR volume. The combination of the alkaline lysis and neutralizing reagents yields a buffer consisting of 20 mM Tris-HCl (pH 8.1) and 0.1 mM EDTA, which is similar to a common DNA storage buffer. We have used this technique to prepare DNA for hundreds of PCR assays and find it remarkably consistent. Based on our initial success, we tested the method with tissues commonly used as a source of mouse genomic DNA. Toes from neonatal mice, 0.2-cm tail snips from weanling mice, ear punches from adult mice and small pieces of spleen (about 25 mg) from adult mice were placed in thermal cycler strip tubes with 75 μL alkaline lysis reagent added to each sample. Samples were heated to 95°C for 10, 20, 30 or 60 min, chilled to 4°C and 75 μL neutralizing reagent were added to each tube. The yield of soluble DNA was measured by a fluorometric assay (1). The performance of HotSHOT DNA in PCR was tested by amplifying a 182bp PCR product and comparing the performance of DNA prepared by proteinase K digestion, followed by phenol:chloroform extraction and ethanol precipitation (3). In this experiment, 1.5 μL HotSHOT DNA was combined with 8.5 μL PCR mixture. Forward and reverse primers were combined at 200 nM each with PCR buffer, 3 mM MgCl2, 0.2 mM each dATP, dCTP, dGTP and dTTP, 0.12 U Taq DNA polymerase (Promega, Madison, WI, USA), in a 10-μL volume. Reactions were covered with 10 μL mineral oil and amplified in a PTC-100 thermal cycler (MJ Research, Watertown, MA, USA) for 40 cycles at 95°C for 40 s and 60°C for 45 s. Following amplification, 10 μL loading dye were added to each well (4 μL 60% sucrose and 1 mM cresol red plus 6 μL water). Twelve microliters of the reaction were loaded on a 12% vertical polyacrylamide mini-gel (8 × 10 cm) and electrophoresed at 165 V for 45 min in 1× TBE buffer (89 mM Tris/89 mM boric acid/2 mM EDTA). Gels were stained for about 2 min in 1× TBE containing 1 μg/mL ethidium bromide and imaged under UV light using an Ambis Imaging System (Scanalytics, Billerica, MA, USA). The yield of soluble DNA varied with the tissue type and heating time (Figure 1). The yield was greatest from spleen. Ear punches, neonatal toes and tail snips produced similar amounts of soluble DNA. The yield of DNA increased with heating time for each of these tissues, most dramatically with spleen. However, the variation in soluble DNA among tissues and heating times had little influence on the amplification of the PCR product (Figure 2). These results suggest that the duration of heating time is not critical to the reaction outBenchmarks