Abstract
The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for an easy extraction and purification of genomic DNA from a wide range of samples, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. RBCs are lysed by dilution into a hypotonic solution. Tissues are broken down and digested by proteinase K in the presence of an anion detergent to release genomic DNA. After precipitation of the detergent and proteins, unique beads that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low concentration samples. No DNA-binding beads or columns are used in the method, eliminating the problem of low yield and the risk of shearing of genomic DNA. The purified samples are free of proteins, lipids, salts, and RNA contamination. Purified samples are also stable for storage and suitable for all downstream applications.
Keywords: blood and tissue, nucleic acid purification, ethanol precipitation
RESULTS
FIGURE 1.
Blood genomic DNA: procedure. TE buffer, 10 mM Tris-Cl, pH 7.5, 1 mM EDTA.
FIGURE 8.
Tissue genomic DNA: results. (a) Genomic DNA was purified from different tissues from an adult mouse. All tissues were dissected, soaked in cell lysis solution, homogenized, digested by adding proteinase K, and incubated in 55°C for 2 h to overnight. (b) Genomic DNA from mouse tissues was digested with EcoRI. The gel shows 75 ng each sample before and after restriction digetions on a 0.7% agarose gel. M, λ/HindIII digest.
FIGURE 2.
DNA yield—different anticoagulants. Genomic DNA from 1 ml human whole blood preserved in different anticoagulants, including sodium citrate, sodium EDTA, citrate phosphate dextrose adenine (CPDA), and sodium heparin, with the method and Puregene's procedure (Qiagen, Valencia, CA, USA). Purified DNA was dissolved in 1 ml TE buffer. The gel shows 10 μl each sample from triplicate preparations loaded on a 0.7% agarose gel. Marker (M), λ/HindIII digest.
FIGURE 3.
DNA yield—fresh versus frozen blood. Genomic DNA from 1 ml human whole blood preserved in sodium EDTA. The results show the average and sd of the total yields for five samples kept in 4°C (fresh) or −20°C (frozen). DNA were quantified using Quant-iT PicoGreen dsDNA assay (Invitrogen, Carlsbad, CA, USA).
FIGURE 4.
DNA yield and quality—different approaches. Genomic DNA from bovine whole blood. DNA from triplicate preparations were combined for analyses. (A) Total yield from 300 μl blood. (B) SDS-PAGE analysis. Proteins in 2 μg purified DNA were precipitated with acetone and resolved on a 4–20% polyacrylamide gel. M, ProSieve protein marker (VWR, West Chester, PA, USA). (C) DNA quality analysis. Each sample (150 ng) was loaded on a 0.7% agarose gel. DNA markers (M), λ/HindIII digest (right) and 1 Kb ladder from Promega (Madison, WI, USA; left). Puregene (P) and the developed methods E1 and E2 are similar solution-based approaches. There is no Advamax beads step in E1 to demonstrate the efficient removal of proteins by using Advamax beads. DNeasy (D) from Qiagen uses a silica gel spin column to capture DNA.
FIGURE 5.
DNA quality—storage stability. Genomic DNA was purified from 1 ml human whole blood with sodium EDTA, stored at −20°C, 4°C, room temperature (RT), or 37°C. The gel shows 200 ng each sample loaded on a 0.7% agarose gel. Marker (M), λ/HindIII digest.
FIGURE 6.
Applications—restriction digestions, PCR. (a) Genomic DNA from human whole blood was digested with selected restriction enzymes. The gel shows digestions of 450 ng DNA loaded on a 0.7% agarose gel. DNA markers (M), λ/HindIII digest and 1 Kb ladder (Promega). (b) Genomic DNA from human whole blood in different anticoagulants was used in PCR amplification of a 0.7-Kb fragment of β-glucuronidase gene. The gel shows 10 μl each PCR product loaded on a 1.5% agarose gel. Marker (M), 1 Kb DNA ladder (Promega).
FIGURE 7.
Tissue genomic DNA: procedure.
SUMMARY
A solution-based method and reagents were developed for purification of genomic DNA from blood samples, tissues, and cells. The protocols are adaptable to various scales and formats.
The approach gives a high DNA recovery efficiency. For low abundant samples, Quick-Precip reagent can be used to enhance DNA precipitation further.
In contrast to conventional, solution-based methods, residual proteins were depleted further by using unique, adsorptive beads to remove contaminants. The supreme purity may lead to ultimate storage, stability, and enhanced consistency in sensitive applications.
The method achieved the highest purity without using DNA-binding columns or beads and achieved greater yield and integrity of genomic DNA.