This study examined the yield and quality of DNA from samples applied to Whatman® FTA® cards, using five common methods of DNA extraction. The data demonstrate that all five methods yield purified DNA of sufficient quantity and quality for both quantitative PCR and short tandem repeat analyses. In addition, EasiCollect™ device for buccal sample collection was shown to be compatible with all five DNA extraction methods, and provided final DNA yields equal to or higher than those from separate foam swabs.
Whatman® FTA® family of products (Figure 1) facilitates collection, transport, purification, and long-term, room-temperature storage of nucleic acids, all in a single device. FTA technology has the ability to lyse cells on contact, denature proteins, remove contaminants, and protect DNA from degradation. In the classic scenario for FTA cards, purified DNA is analyzed while still bound to the matrix (i.e., using the “punch-in” method). This study examined an alternative option for FTA cards—DNA extraction into solution.
Whole blood and buccal cells were collected onto FTA cards.
Blood from ten unrelated individuals was collected by venipuncture into vacuum collection tubes containing dipotassium EDTA. Blood (40 μL) was applied to each FTA Micro Card. Cards were dried for at least 3 h at room temperature.
Figure 1. Whatman® FTA® family of products.
Buccal samples were collected from ten unrelated individuals, using either EasiCollect devices from Cytiva (Figure 2) or foam swabs. Samples were applied to FTA cards according to the manufacturer’s instructions. Cards were dried for at least 2 h at room temperature.
Figure 2. EasiCollect™ is an all-in-one device that collects buccal samples and transfers them to an integrated FTA card.
DNA was extracted or isolated from FTA cards using five different methods, exactly as described in the following protocols. These methods include:
Prepare extraction buffer and Proteinase K stock solution. Extraction buffer: 10 mM Tris-HCl, pH 8.0; 10 mM EDTA, disodium salt, pH 8.0; 100 mM sodium chloride; and 2% v/v SDS. Proteinase K stock solution: 20 mg/mL in sterile distilled water.
Prepare a 5% w/v suspension of Chelex 100 resin in sterile water prior to use; stir to maintain a suspension.
Reagents and columns are provided with the kit.
Reagents and columns are provided with the kit. Phosphate-buffered saline (PBS) is also required.
Reagents and columns are provided with the kit. A magnetic separation stand is also required.
DNA extracted from FTA cards was measured using quantitative PCR (qPCR). DNA was subjected to short tandem repeat (STR) analysis to assess its quality.
Extracted DNA was measured using quantitative PCR (qPCR) and an ABI™ 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Reactions were prepared using the Quantifiler™ Human DNA Quantification Kit (4343895, Applied Biosystems). The DNA quantitation assay combines two 5’ nuclease assays. The first is a target-specific human DNA assay consisting of two primers for amplifying human DNA and one TaqMan™ MGB probe labeled with FAM™ dye for detecting the amplified sequence. The second assay is an internal PCR control (IPC) assay consisting of two primers for amplifying the IPC template DNA and one TaqMan MGB probe labeled with VIC™ dye for detecting the amplified IPC DNA. Standard curves for DNA quantitation were prepared using control DNA supplied with the kit.
STR analysis of extracted DNA was conducted using the Powerplex™ 16 System (Promega) according to the manufacturer’s instructions. Amplification was performed in a 9700 thermal cycler (Applied Biosystems), and amplification products were analyzed on an ABI PRISM™ 3130XL Genetic Analyzer (Applied Biosystems) according to the manufacturer’s instructions.
DNA quantitation by qPCR
DNA was extracted from FTA discs containing blood or buccal cells from ten unrelated individuals. Four sizes of disc were used for each extraction method, and the total quantity of extracted DNA per disc was determined as described in Materials and Methods.
For each of the five extraction methods, a minimum of 10 ng of genomic DNA was recovered from blood samples on FTA cards (Figure 3). The amount of DNA extracted using each method increased as the disc size increased.
Figure 3. Total DNA (ng) isolated from discs taken from the center of FTA cards spotted with 40 μL of whole human blood. Each point on the graph represents the mean recovery of DNA from a single disc from ten individuals. Error bars represent standard deviations. Note that the y axis represents a logarithmic scale.
Buccal samples have historically produced greater variability in DNA yield than blood samples. The variability in buccal DNA yield is most likely due to clumping of cells on the matrix. As for blood samples, the amount of DNA recovered with each extraction method tended to increase as the disc size increased. Note that higher DNA yields were often obtained from buccal samples collected with EasiCollect devices than from samples collected with foam swabs (Figure 4). The data shown in Figures 3 and 4 illustrate that commonlyused DNA extraction methods and kits do provide DNA of sufficient quantity and quality for successful qPCR.
DNA extracted from collected buccal samples
Figure 4. Total DNA (ng) isolated from discs taken from the center of an FTA card containing buccal cells, applied with either a foam applicator or an EasiCollect device as indicated,
Figure 5. A-O. STR profiles of DNA extracted from FTA cards containing blood or buccal samples from Test Subject 1.
STR data were generated to determine the accuracy of allele calls for DNA extracted from FTA cards. For each DNA extraction method, duplicates of DNA from FTA cards were run for three blood samples, three buccal samples collected using EasiCollect devices, and three buccal samples collected using separate foam swabs, resulting in a total of 18 samples per DNA extraction method. Note that the three samples were from three test subjects. Of the 90 STR profiles collected, 87 samples provided quality data with accurate allele calls on the first attempt; three samples required a reinjection for 100% accurate allele calls. These data illustrate that commonly-used DNA extraction methods and kits provide DNA of sufficient quantity and quality to support allele call accuracy as high as 100% in STR analysis.
This study shows that five common DNA extraction methods yielded enough DNA from FTA cards for downstream quantitative analysis. This DNA was of sufficient quality for determining human identity via STR profiling. In addition, collection of buccal cells using the EasiCollect device often resulted in a higher yield of DNA than collection using a foam swab. These data demonstrate that the EasiCollect device is compatible with current validated laboratory protocols for DNA extraction.