Compositions and Methods for Nucleic Acid Extraction from Biological Samples
a biological sample and composition technology, applied in the direction of nucleic acid reduction, microorganisms, biochemistry apparatus and processes, etc., can solve the problems of less or no amplification, lack of effective extraction from the sample, and it is more difficult to extract nucleic acids from tissues than to extract nucleic acids from cells, so as to facilitate extraction
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example 1
[0046] This example illustrates the extraction of DNA from mammalian tissue samples, Drosophila samples, C. elegans samples and from plant seed samples.
[0047] The extraction method uses an extraction solution, a tissue preparation solution and a neutralization solution. The Extraction Solution (referenced herein as E7526) contains 0.25 M KCl, 0.01 M EDTA and 0.1 M Tris-HCl, at a pH of 9.5. The Sample Preparation Solution contains Proteainase K from Tritirachium Album Libium in an aqueous solution (˜800 Units / mL) containing 40% glycerol, 1 mM calcium acetate and 10 mM Tris-HCl at pH 7.5. The Neutralization Solution B contains 3% bovine albumin and 10 ppm Kathon.
[0048] The following procedures are carried out for extraction of DNA from mammalian, drosophila or C. elegans samples at room temperature and from seed samples at 55° C.
[0049] A. DNA extraction from Mouse Tails, Animal Tissues, Hair, Saliva, Drosophila or C. Elegans.
[0050] 100 μl of Extraction Solution is placed into a mi...
example 2
[0080] This example illustrates the extraction of DNA by the present method using tissues from mouse ear punches and tail snips and compares extraction with earlier methods (Chen et al.,1990, supra; Ren et al., 2001, supra).
[0081] All materials were obtained from Sigma-Aldrich (St. Louis, Mo.) unless otherwise noted. PCR primers were obtained from SigmaGenosys (The Woodlands, Tex.).
[0082] Tissue samples obtained were mouse ear punches (⅛ inch disc) and mouse tails (0.5 cm tail tip and next 0.5 cm section up from that tip). The tail pieces were either fresh or frozen from a previous tissue collection which had been stored at−20° C. Each extraction was setup by placing 1 mouse ear punch or mouse tail into a 1.5 ml microcentrifuge tube. Extraction using the present method was performed as described in Example 1. Comparative extractions were performed using the methods of Chen et al. (1990, supra) and Ren et al. (2001, supra).
[0083] Two tubes were prepared for the extractions of each...
example 3
[0095] This example illustrates the effect of pH and KCl concentration on extraction of DNA from tissue samples.
[0096] Frozen mouse tail cuttings were extracted and amplified as described in Example 2. Results are shown in FIG. 1, Rows III and IV.
[0097] PCR amplification following extraction at pH adjusted to pH 10.5 (lanes 1 and 2), adjusted to pH 9.5 (lanes 3 and 4), adjusted to pH 8.5 (lanes 5 and 6) and adjusted to pH 7.5 (lanes 7 and 8) with negative and positive controls in lanes 9 and 10. As can be seen in the figure, little difference in the extraction efficiency was seen from pH 8.5 to pH 10.5. At pH 7.5 one of the two preparations did not show PCR amplification product which suggests that extraction efficiency might begin to decrease as pH decreases to 7.5 and below.
[0098] KCl concentration did not show any effect on extraction efficiency at 200 mM KCl (lanes 1 and 2), at 100 mM KCl (lanes 5 and 6), at 50 mM KCl (lanes 9 and 10) or at 0 mM KCl (lanes 13 and 14). Lanes 3...
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