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Buccal Cell DNA Extraction

Genetic research has recently seen a marked increase as interest in understanding the genetic basis of diseases and drug regimens increases. Almost all of these studies require DNA isolations. Blood samples, in particular white blood cells, are an excellent source to obtain large amounts of genomic DNA. However, because of the invasiveness and cost of obtaining and testing blood samples, alternative methods are needed particularly in epidemiologic studies. Buccal cells provide a feasible and noninvasive approach to supply DNA for genetic testing.

There are two types of methods for collecting buccal cells: dry and wet procedures. Dry procedures use cytobrushes, buccal swabs, or other methods to scrape the oral mucosa. Wet procedures include swishing liquids in the mouth and spitting them into a collecting vessel.

The swish method yields a higher amount and longer fragments of DNA, however, it requires more steps, is more cumbersome, and is associated with an increased cost. Dry methods are simpler and more feasible for large-scale mailings; additionally, they have a lighter weight and are more cost effective. Recent studies have shown similar successful DNA amplification and polymerase chain reaction (PCR) applications from both procedures. Protocols to extract DNA by dry procedures include the use of chemicals such as phenol-chloroform, NaOH, or already prepared kits containing all the components and a manual for DNA extraction steps. Studies comparing the yield and purity of the obtained DNA among different methods of extraction are needed.

Using NaOH is less expensive than marketed kits. However, the significance of the difference in DNA yield, purity and quality between NaOH and marketed kits needs to be determined. The primary objective of this study was to compare buccal cell DNA yield and purity extracted by two different techniques, namely, NaOH protocol or Nucleospin Tissue® prepared kit (BD Biosciences, Macery-Nagel, Germany) using a dry DNA collection method (cotton swabs). Quantity, feasibility, and time consumption were assessed, as well. Instructions for the collection of DNA using cotton swabs (Nuova Aptaca™, Canelli, Italy) were given for each subject enrolled in the study by the experimenters. The subjects abstained from smoking, drinking, and/or eating for 45 min before sample collection. The subjects were asked to twirl a sterile cotton swab on each inner cheek for 15 sec. The swabs were returned to the laboratory. DNA was extracted from the cotton swabs within 24 hr. All samples were processed at room temperature (24°C) in accordance with good laboratory practices.

DNA extraction

NaOH protocol. DNA was extracted according to a protocol using NaOH in 150 samples. The swabs were separated from the sticks with scissors and they were transferred to 1.5 ml EpendrofTM tubes (Ismaning, Germany). Three hundred microliters of NaOH 50 mM were added to each tube. The tubes were closed and vortexed for 10 sec. They were placed in a thermomixer for 5 min at 95°C. Then, the swabs were removed and discarded. Thirty microliters of 1 M Tris HCl, pH  8.0 was added to each tube. The tubes were centrifuged at 13,000 rpm for 2 min. The supernatant (DNA) in each tube was used for analysis. Nucleospin protocol. Nucleospin Tissue® prepared kit was used to extract genomic DNA from the other 150 samples following the Nucleospin Tissue® kit manual instructions (Nucleospin ® Tissue Kit manual, 2004).

In brief, the dry swab was placed in 2-ml microcentrifuge tubes. Phosphate buffered saline (PBS), 400 ml, was added with 25 l proteinase K solution to the swabs. This step aimed at prelysing the sample. The lysis step involved the addition of 400 l of a prepared buffer to the previous solution. Vigourous vortexing was needed and the samples were incubated at 70°C for 10 min. Four hundred microliters of 98% ethanol was added to each sample and revortexed, thus adjusting DNA binding conditions. To bind DNA, 600 l of the samples were centrifuged at 11,000g for 1 min. The next step was to wash the silica membrane with a wash buffer and centrifuge for 1 min at 11,000g. A second wash with another buffer was done and centrifuged again. To dry the silica membrane, the samples were centrifuged one more time (residual ethanol was removed during this step). To elute highly pure DNA, 100 l of a prewarmed elution buffer (70°C) was used and the samples were incubated at room temperature for 1 min. Finally, the samples were centrifuged for 1 min at 11,000g once more. Obtained DNA was collected.

Yield and purity determination

Quartz cuvettes were used to read the absorbance at a wavelength of 260 nm and 280 nm using spectrophotometry. DNA concentration was calculated by multiplying the absorbance at 260 nm by 50 (1 unit is g/ml). A 260:A280 ratio was calculated to check for DNA purity. To validate DNA quality and quantity, angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) gene polymorphisms were amplified from the extracted DNA by either NaOH or Nucleospin Tissue Kit® using the PCR as described by Saab et al. (2007). The amplicons were separated and sized by electrophoresis on a 2.5% agarose gel and visualized directly with ethidium bromide staining. The quality of the DNA from samples was assessed with the insertion allele manifested as a 490 bp and the deletion allele visualized as a 190 bp band.

The cost of extracting DNA per sample from the cotton swabs using NaOH and Nucleospin Tissue® kit was calculated. The cost was based on actual charges for equipment and supplies used in the study for both groups; i.e., the price of the kits ordered from the supplier and the materials and chemicals used in the NaOH method. The cotton-swabs cost was not included in the study for the same number and type of swabs were used in both groups. Cost of the personnel time for processing the samples was not considered, as well. The average cost per sample in USD was 0.06 and 3.08 for NaOH and Nucleospin Tissue ® kit, respectively.

The average time consumption per sample for NaOH vs Nucleospin Tissue® kit is presented in Table 1. The elapsed time was recorded for 12 samples processed together of each group, then calculated per sample. The average time for the processing of 12 samples was 16 min for the NaOH group and 43 min for the Nucleospin Tissue® group. Time consumption using NaOH protocol is significantly less than the time needed to extract DNA by the Nucleospin® Tissue kit. The role of pharmacogenetics is going to have an increased emphasis especially in the development and prevention of cancer and other diseases. Thus, cost-effective methods of DNA collection and extraction need to be determined. The aim of our study was to evaluate NaOH versus Nucleospin Tissue® kit for DNA extraction collected from buccal cells, and then to determine a simple method for DNA extraction.

The two methods were compared and both the quantity and purity of DNA and the feasibility and cost of the procedures were assessed. Many studies have shown that the collection of DNA with cytobrushes using simple instructions is cost effective in largescale studies and yields sufficient quantity and good quality of DNA for genotyping. A total of 15.8 g/ml of DNA from cytobrushes and 12 g/ml from mouthwash were obtained. The collection of human genomic DNA from buccal cells for genetic studies using cytobrush, mouthwash, and treated cards was compared. The mean DNA yield was found to be 3.5, 4, and 2.6 g/ml for cytobrushes, mouthwashes, and treated cards, respectively. The results confirmed that the protocol based on cytobrushes should be preferentially used because of its ease of collection, good DNA quality and sufficient DNA quantity. Another study determined the long-term stability, quantity, and quality of genomic DNA collected by the mouthwash method in order to use it in pharmacogenomic studies.

A median of 12.6 g/ml and 13.6 g/ml of DNA extracted by NaOH and phenolchloroform, respectively, using cytobrushes was obtained in an epidemiologic study (Garcia-Closas et al., 2001). DNA yield obtained in our study (14.1 g/ml for NaOH versus 16.6 g/ml for the kit) was higher than previously reported studies using buccal cells for DNA extraction. DNA extracted in this study from buccal cells using cotton swabs provides adequate amount of DNA for further usage. Both methods (NaOH and Nucleospin Tissue® Kit) used supplied sufficient DNA yields. A mean A260:A280 ratio was 1.8 and 1.9 for the brush and mouthwash, respectively, in a large-scale study. PCR success rate was 100% for all samples. DNA purity from another study resulted in ratios of 1.6, 1.7, and 1.1 for cytobrush, mouthwash, and treated cards, respectively.

DNA ratios of 1.9 and 1.3 for phenol-chloroform and NaOH, respectively, for cytobrushes were obtained in an epidemiologic study. Samples collected by mouthwash had ratios of 1.9 for both phenol-chloroform and PureGene Kit (Gentra Systems, Minneapolis, MN). In this study, a mean ratio of 1.08 and 1.245 were obtained using NaOH and Nucleospin Kit®, respectively. Comparing DNA purity with the NaOH method and the Nucleospin Tissue® Kit revealed a slight insignificant difference. Although the samples are not 100% pure (ratio  1.8), this will not limit the DNA usage for further applications; PCR amplification was successful for assessed samples by either method. In terms of cost, the relevant studies compared different methods of DNA extraction. In one study, the estimated cost per person for buccal brush DNA was $8.5 and $18 for the mouthwash.

All of the samples were extracted using QIAamp Mini Kits (Quiagen, Hilden, Germany). The final cost in another study was per sample: $4.66, $7.17, and $2.81 for cytobrush, mouthwash, and treated cards, respectively (Mulot et al., 2005). The cost per sample of extracting DNA by using NaOH ($0.06 per sample) is more than 50 times less expensive than the Nucleospin Tissue® Kit ($3.80 per sample) and much less expensive than in reported studies. Given the comparable yield and purity between the two methods, the NaOH protocol for DNA extraction provides a cost-effective approach. As for collection time, 1.7 min was needed for DNA extraction per sample step for either cytobrush or mouthwash in a human genomic DNA collection study (Mulot et al., 2005). Personnel time for DNA extraction using the Nucleospin Tissue Kit (3.59 min per sample) is more than double the time needed for extraction following the NaOH protocol (1.33 min per sample). This also favors NaOH as being a time saving alternative to Nucleospin Tissue® Kit.

In summary, DNA yield and purity are comparable whether DNA was extracted by NaOH protocol or Nucleospin Tissue® Kit. NaOH protocol is less expensive and less time consuming, therefore, it provides a more feasible and cost-effective alternative for DNA extraction. We recommend using NaOH protocol for DNA extraction in large-scale studies. Further studies are needed to compare DNA extracted by different chemical methods versus other marketed kits.


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This entry was posted on Tuesday, January 22nd, 2008 at 7:38 pm and is filed under Forensic DNA. You can follow any responses to this entry through the RSS 2.0 feed. Responses are currently closed, but you can trackback from your own site.