The PUREGENE™ DNA Isolation kit (Gentra Systems Minneapolis, MN) is used to extract DNA from mummified tissues.[1,10]
In this kit, purification of genomic DNA is carried out using ammonium acetate as a substitute for toxic organic solvents in the protein precipitation step. The pulverized tissue is mixed with cell lysis solution containing Tris-HCl, ethylenediaminetetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS), and incubated at 65°C for 60 min. After proteinase K solution (20 mg/mL) was added to the lysate, incubation at 55°C is carried out until tissue particulates have dissolved. RNase A solution (4 mg/mL) is added to the lysate and incubated at 37°C for 60 min. Protein precipitation solution (ammonium acetate) is added to the solution and vortexed vigorously then centrifuged. The DNA is precipitated with 100% iso-propanol (2-propanol). After centrifugation, the pellet is washed several times with 70% ethanol and then air-dried. Purification is carried out with a Microcon™ YM-100 microconcentrator ( Millipore, Bedford, MA). Finally, the DNA is hydrated with sterile distilled water. The extract is quality-checked by agarose gel electrophoresis and determined by a UV spectrophotometer. The £260/£280 ratio is recommended to be greater than 1.5, as the success rate of PCR is thought to depend on the purity. When the DNA yield is expected to be low (<1 mg), DNA carrier such as glycogen solution (20 mg/mL) is added.
Method 2 (Organic Solvents Method: Phenol/Chloroform Extraction)
This method has been used conventionally and is still widely used for degraded and aged samples. Many modifications are presented for the kind of organic solvents or reagent of digestion and so on. Here the representative method is described as applied to the extraction of mummified tissues.
Guanidium thiocyanate (GTC) is a chaotropic agent commonly used for tissue lysis of archived specimens.[6,7] After homogenization, the pellets are lysed in a 5 M GTC buffer containing 5 M GTC, 0.5% bovine serum albumin, 80 mM EDTA, 400 mM Tris-HCl (pH 7.5), and 0.5% sodium-N-lauroylsarcosine at 60°C for 1 hr and then at 37°C overnight. DNA is extracted twice with phenolchloroform at a 1:1 ratio, followed by chloroform once, and then precipitated by the addition of a 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. The pellets are washed with 70% ethanol and air-dried. They are then dissolved in Tris-EDTA [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] buffer.
Samples are put into digestion buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM NaCl, 2% SDS) containing proteinase K solution (10 mg/mL) and incubated for several hours at 56°C.[1,8] Additional aliquots of proteinase K are added to achieve complete digestion. Next, the solution is extracted with phenol/chloroform/isoamyl alcohol (25:24:1) by a mild mixer. After centrifugation, the upper phase is transferred to a new tube. The extraction is repeated until the interface is clear. Finally, one more extraction is performed with chloroform to remove phenol, and the aqueous phase is put into another tube. After the addition of 5 M NaCl solution, DNA is precipitated with 100% ethanol. The recovered DNA is washed three times with 70% ethanol and air-dried. It is hydrated with sterile distilled water.
Microconcentrator-Based Method. Samples are put into the solution containing 10 mM Tris-HCl (pH 8.0), 2 mM EDTA, and 10 mM NaCl.[2,8,11] The tissue is broken up into small pieces by vortexing, and then collagenase is added. The tubes are incubated at 37°C with slow agitation for 3 hr. Sodium dodecyl sulfate is then added as well as dithiothreitol (DTT) and proteinase K. Incubation is continued for approximately 20 hr. The solution is extracted twice with water-saturated phenol of neutral pH and once with chloroform/isoamyl alcohol (24:1). It was concentrated by a Centricon 30 microconcentrator.
Silica-Based Method. Extracts of DNA are made from each sample by a silica-based method that is highly efficient in the retrieval of ancient DNA.[5,12]
DNA extraction is carried out with the supernatants in an automated nucleic acid extractor, starting with proteinase K digestion at 56°C for 1 hr. A standard phenol/ chloroform extraction is carried out, followed by mixing the samples with a silica powder (glass milk, Dianova). This process produces a binding of DNA to the glass beads in the presence of isopropanol and sodium acetate (2.0 M, pH 4.5). In the last phase of the automated extraction procedure the DNA/glass milk samples are collected on filtration membranes and washed with ethanol. Finally, the DNA is manually eluted from the silica beads with sterile water and run on a 3% agarose gel.
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