Development and validation of the high-quality 'rapid method for swab' to genotype the HTTLPR serotonin transporter (SLC6A4) promoter polymorphism

Abstract

Background: The importance of genetic variation to the etiology of neuropsychiatric disorders is well established and is currently being examined for diagnosis and treatment. The most popular method of obtaining material for genotype analysis, high-yielding DNA extraction from blood, has several limitations, including invasiveness, need for skilled individuals to collect material, and requirement for cold storage. Saliva sampling is noninvasive and trained personnel are less necessary, but it still requires a relatively high level of subject compliance. Buccal mucosa cells sampling is almost completely noninvasive, reducing compliance issues significantly. Samples collected have been shown to produce usable DNA after shipment through conventional mail. The DNA produced by rapid elution of these swabs in chaotropic buffers is, however, of limited quality and low purity.

Objective: Our aim was to develop a rapid, economical, and environmentally safe method for extraction of high-quality genomic DNA, which can be used to determine clinically important genotypes from trace quantity samples and which has sufficient yield for multiple assays.

Methods: We developed a method of extracting high-quality genomic DNA from buccal swab, which we termed the 'rapid method for swab' (RMS). We compared RMS with two established procedures, specifically the original rapid method and the commercially available Buccal Amp method. We assessed the generated genomic DNAs by their (i) quality, (ii) quantity, (iii) restriction enzyme digestibility, and (iv) PCR-based genotyping in addition to time, cost, and environmental impact of the procedures.

Main results: DNA generated by RMS was of higher purity than that by Buccal Amp. RMS is nonenzymatic and does not use strong chaotropic salts or extreme pH. We also showed the suitability of RMS-DNA for LA/LG genotyping as generated by PCR using 7-deaza-dGTP.

Conclusion: The RMS procedure is novel, efficient, safe, and yields sufficient material for multiple genotyping analyses. The RMS produces DNA of high quality from a single human buccal swab. RMS is a noninvasive technique and particularly suitable for children and older individuals and in field collection settings.

Fig. 1. Assessment of the quality of DNA generated by different extraction methods

A. A volume of 20 μl of prepared DNA was run on 1% agarose gel, either uncut (lanes 3, 5, 7, 9) or after digestion with EcoRI (lanes 4, 6, 8, 10). Lane 1: commercial 1kb DNA ladder; lanes 3-4: commercially purchased DNA. lanes 5-6: DNA prepared by BuccalAmp kit; lanes 7-8: DNA prepared by the rapid method (RM); lanes 9-10; DNA prepared by the modified rapid method (RMS) B. samples of DNA purified by RM or by RMS with different buffer conditions were run on 1% agarose gel or digested with EcoRI and run on agarose gel. Lane 1: 500bp DNA ladder; lanes 2-7: undigested DNA; lanes 8-13: DNA digested with EcoRI. DNA was visible for commercial DNA (lanes 2, 8), and for RMS DNA prepared at buffer pH of 5.4 (lanes 3, 9), 7.6 (lanes 4, 10), and 10 (lanes 5, 11). Preparations from the RMS method adjusted by NaOH (lanes 6, 7, 12, 13) were also run.

Fig. 2. Spectral analysis of DNA extracted by different methods

DNA was extracted from buccal swabs by ten different methods as described in the text. A spectrum for 100μl of each was generated at 220nm to 300 nm with a Beckman DU-70 spectrophotometer. On each spectrum, the position of OD260 is indicated with a dashed line. The major peak is indicated with an arrow. Samples analyzed were as follows: A. Commercial DNA sample, 100ng diluted into 100μl. B. Rapid Method (RM), C-H. Modified rapid method (RMS) with different buffer conditions, specifically pH 5.4 (C), pH 7.6 (D), pH 10 (E), 50mM NaOH (F), and 50mM NaOH equilibrated with 1M Tris pH 7.6 (G), Epicentre buffer used with RMS (H). I. The conventional Epicentre BuccalAmp method. J. TKM buffer used with the BuccalAmp `heat and vortex' method.

Fig. 3. HTTLPR genotyping PCR of DNA generated by different extraction methods

HTTLPR genotyping PCR was carried out on DNA samples generated by different extraction/purification methods as described in the text. A volume of 9μl of each sample was used. DNA bands associated with the `s' and `l' alleles and the `additional' band associated with the `s/l' heterozygote are indicated. A: Lane 1: 100bp ladder; lanes 2-10: DNA prepared by the RM (2), RMS (3-7), Epicentre BuccalAmp (8), Epicentre buffer followed by RMS (9), or TKM followed by BuccalAmp heat and vortex method (10). B: Genotyping PCR carried out on additional lab samples to verify heterozygote in RMS DNA. Lane 1: 100bp ladder; lane 2 empty; lane 3: DNA prepared by RM; lanes 4-5: DNA prepared by RMS, pH 7.6 buffer. Positions of the `s' and `l' allele bands are indicated. C: Genotyping PCR carried out in parallel on DNA extracted from swabs via RMS and from whole blood via RM. Lane 1: 100bp ladder; 2, 5: DNA from volunteer `A'; 3, 6: DNA from volunteer `B'; 7, 9: DNA from volunteer `C'. Lanes 2-4 were prepared from buccal swab by RMS. Lanes 5-7 were prepared from whole blood by RM. Genotypes are indicated.

Fig. 4. Presence of `additional' (~900bp) band in TAE-PAGE of HTTLPR heterozygotes prepared by BuccalAmp method

Multiple different human DNA samples were prepared by BuccalAmp method as described previously (Hayden et al., 2007). Samples were run on 4.5% native TAE-PAGE. Genotypes of `s/s', `l/l', and `s/l' appeared in the sample. An `additional' band of approximately 900bp appeared in all and was unique to `s/l' samples.

Fig. 5. Determination of the nature of a high molecular weight `band' by native acrylamide and agarose electrophoresis

Heterozygous DNA was used for HTTLPR genotyping and subject to electrophoresis on `A'; nondenaturing TAE-PAGE, `B'; TAE agarose. An approximately 900bp band appeared only on the native TAE-PAGE gel. This band did not appear on the agarose gel.

Fig. 6. HTTLPR `l' and variant illustrating location of HpaII sites and the L_A/L_G site

A. Location of the HTTLPR polymorphic region in relationship to the +1 transcription start site of the SLC6A4 gene, diagram based on published sequences (Lesch et al., 1994) and Nakamura et al (Nakamura et al., 2000). Sequences of multiple `l' variant alleles (Nakamura et al., 2000) were analyzed for the presence of HpaII sites. The L_G polymorphic site (which creates an additional HpaII site) is also indicated.

Fig. 7. Use of RMS-prepared DNA for HTTLPR L_A/L_G screening

HTTLPR genotyping PCR was carried out on DNA samples generated by RMS as described in the text. A volume of 9μl of sample previously determined to be l/l genotype was used. PCR was followed by overnight (16hr) digestion of reaction products with HpaII restriction enzyme. Both full and partial digestion products were present. Sample was L_A/L_A, since none of the potential L_G digestion products (dashed-line arrows) appeared.