Gelatin-coated magnetic nanoparticles-based DNA isolation method: A comparison with commercial DNA isolation kits from whole blood

DNA isolation is the first step for most molecular tests, thus indicating the importance of using a reliable and suitable method for this procedure. The use of magnetic nanoparticles for blood DNA isolation offers several advantages including subjecting little mechanical stress to your sample, obtaining higher quality and quantity of DNA, and non-laborious procedures. In this study, we isolated genomic DNA using gelatin-coated magnetic nanoparticles from blood samples and test its use compare with three commercial blood DNA isolation kits. DNA purity and yield were assessed by measuring absorbance at A260/A280 and by agarose gel electrophoresis. The suitability of the isolated DNA for downstream applications was analyzed by endpoint PCR and Sanger sequencing. In this study, gelatin-coated magnetite particles for genomic DNA isolation provide an efficient, simple, and inexpensive method that does not require the use of commercial blood DNA Isolation Kit.


INTRODUCTION
An appropriate DNA isolation method for a specific biological sample (whole blood, saliva, stool, urine, fresh tissue, or paraffin-embedded tissue) is a prerequisite for any molecular testing. This selection is dependent on many factors including yield, purity, time, safety, specialized equipment requirement, trained personnel, intended downstream applications, cost, and sample source.
Blood is an ideal source of human genomic DNA (Cho et al., 2007). However, isolating genomic DNA by traditional methods is a time-consuming process, and phenol and chloroform are toxic reagents that endanger health (Hansen et al., 2013).
Further, traditional methods, such as phenol isolation, isopropanol precipitation, formamide lysate method, nonorganic solvent isolation, and glass particle adsorption, are ineffective for isolating genomic DNA from a trace, dried, and frozen blood. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA (He et al., 2013). Saliva samples are a good alternative source of genomic DNA owing to the painless and noninvasive collection (Bux et al., 1995).
Compared to blood, saliva samples are much more convenient, efficient, and accessible if DNA can be isolated and purified.
A need for a reliable, easy to use, low cost, and not requiring special equipment DNA isolation method let us develop a DNA isolation kit for blood biological sample. The use of magnetic nanoparticles provides several advantages in biotechnological applications such as enzyme purifications, immunoassays, immunotherapy, and nucleic acid isolation (Borlido et al., 2013;Intorasoot et al., 2009) Even though uncoated magnetic nanoparticles can bind to DNA and can be used for its isolation, polymer-coated magnetite nanoparticles provide a higher recovery of DNA. Magnetic nanoparticles are coated with different polymers such as agarose and silica (Taylor et al., 2000;Yoza et al., 2002;Chiang et al., 2005) previously described and used for bacterial cells. In this study, we compare three commercial DNA isolation kits with a gelatin-coated magnetic nanoparticles-based method to isolate blood biological samples.

Materials
The following materials were used: gelatin-coated magnetic nanoparticle (GMNPs) genomic DNA isolation method called INBIOMag Genomic DNA Kit (INBIOMEDIC, Peru), QIAamp DNA Blood Mini Kit (Qiagen Cat. No. 51104); GeneJET Genomic DNA Purification Mini Kit (Thermo Scientific, Cat No. K0721) and MagJET Genomic DNA Kit (Thermo Scientific, Cat No. K2721). Oligonucleotide primers were synthesized by Macrogen Inc., Korea. All other chemicals and enzymes used were of high-grade purity.

Sample Collection
Peripheral venous blood samples were collected from 30 participants, who visited INBIOMEDIC Research and Technological Center. After obtaining written informed consent, 3 ml of blood samples were collected in a tube containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Each sample was divided into 1 ml aliquots and stored at −20 °C until DNA isolation. DNA isolation from all samples was completed within 3 days after collected blood. The study protocol was approved by INMENSA Ethics Committee, Peru.

DNA extraction methods
The four methods are described: A: Gelatin-coated magnetic nanoparticle genomic DNA isolation method (INBIOMag Genomic DNA Kit): 400 ul of whole blood was used and mix with 1.2 ml of Red Blood Cell lysis (RBC) buffer by a vortex. The solution was incubated at room temperature for 2 min and centrifuge 13000 g for 1 min. Pellet was mixed with RBC buffer and centrifuge 13000 g for 1 min. Pellet was mixed with 100 ul of NaCl Solution and 40 ul of Proteinase K, then it was added 600ul of cell lysis buffer and incubate to 56 °C for 10 min. After incubation, 50ul of GMNPs (50 mg/ml) with 400 isopropanol and 400 ul of binding buffer (1.25 M sodium chloride and 10% polyethylene glycol 6000) were added to the lysate. The solution was mixed and incubated at room temperature for 10 min. The magnetic pellet was immobilized in a magnetic rack and the supernatant was removed. The magnetic pellet was washed three times with wash buffer and dried for 15min. The magnetic pellet was then resuspended in 100 ul of Tris-EDTA (TE) buffer and incubated at 65°C for 10 min. The supernatant containing the DNA and was transferred to a fresh tube. D. MagJET Genomic DNA Kit: 3 volumes of the sample was mixed with 1X RBC Buffer and incubated on ice for 4-7 minutes. The solution was centrifuge in cold at 7000 g for 5 min. The supernatant was removed. The remainder of the extraction procedure was carried out according to the manufacturer's protocol.
The yield and quality of the DNA isolation methods were analyzed by QIAexpert.

Gel electrophoresis score
To observe possible degradation due to the action of nucleases during the isolation procedure, 1 μg of stock DNA from samples representative of each isolation method was electrophoresed on 1% agarose gel and visualized with blue light. A smeared band was indicative of sheared or degraded DNA samples.

PCR amplification
Genomic DNA isolation quality from whole blood was analyzed by PCR amplification of the BRCA-1 gene. All PCRs were performed in a 50 ul reaction volume using Platinum Taq Polymerase (Invitrogen) according to the manufacturer´s procedure and using the GTQ cycler 24 (Hain).

DNA sequencing
PCR products obtained from genomic DNA extracted from whole blood, were purified and sent for sequencing (Macrogen) using the primers BR1c68_69delF and BR1c68_69delR.

DNA concentration and yield
The total concentration and yield of isolated DNA samples were estimated from spectrometric measurements using the QIAexpert platform. Summary statistics of 30 samples isolated using four methods are shown in Table 1. Method D extracted the most concentrated DNA with an average of 53.90 ng/μl, followed by method A with an average of 36.78 ng/μl, both are methodologies based on magnetic nanoparticles.
Method B and C showed a median value of 16.03 and 10 ng/μl respectively, both are methodologies based on silica columns. The total yield of isolated DNA from each sample was calculated by multiplying DNA concentration with the final elution volume of 100 μl. All samples isolated using methods A and D had a total DNA yield above 2ug required for sequencing.

DNA purity
To assess DNA isolated purity by four different methods, absorbance was measured at 230, 260, and 280 nm wavelengths and the ratio of these absorbances were computed to estimate the relative purity of test samples. A260/A280 and A260/A230 ratios are summarized in Table 2. Methods A, B, and D isolated the purest DNA from 30 samples with a mean A260/A280 ratio of 1.62, 1.70, and 1.72, respectively. Method C had the lowest mean A260/A280 ratio of 0.94.
The results of the A260/A230 ratio showed that methods A, C, and D had mean values >1.0 which was indicative of pure DNA free of organic contaminants (Table 2). Method B however, gave values lower than 1, which suggested the presence of residual phenol or chaotropic salts that strongly absorb at 230 nm.

DNA integrity
DNA integrity was checked by electrophoresis of stock DNA samples representative of four isolation methods, as shown in Figure 1. All four isolation methods were capable of isolating non-degraded, slow-migrating genomic DNA with high-molecularweight >10 kb. A method sample showed the least amount of shearing compared to B, C, and D methods samples.

End-point assay: PCR
BRCA-1 amplification by PCR was followed by the separation of PCR products on 1.5 % agarose gel. As shown in Figure 2, the amplification pattern was consistent for all samples using method A and was further confirmed by direct sequencing.

DNA sequencing
Sanger sequencing was performed on 5 DNAs samples isolated by A method. The alignment in Figure 3 showed the successful detection of candidate mutations for each sample. The sequencing performance in the DNA isolated from the Gelatincoated magnetic nanoparticle genomic DNA method is optimal. Gel electrophoresis analysis of PCR products isolated from human whole blood using method A in five samples. Molecular weight marker: 1 kb Plus DNA Ladder

Assessment of time, cost, and labor intensity
Other desirable factors considered for routine DNA isolation are the time involved, cost incurred, and the intensity of labor that the operator must put in. The fastest isolation protocol was the column-based method B followed by method C. Method A and D were the most time-consuming procedures. Relatively, method A was the cheapest since it did not require the use of complex equipment.

CONCLUSION
In summary, the gelatin-coated magnetite particles for genomic DNA isolation method (INBIOMag Genomic DNA Kit) developed in this study provide an efficient, simple, and inexpensive method that does not require the use of commercial DNA Isolation Kit.