A Simple Method to Extract DNA from Hair Shafts Using Enzymatic Laundry Powder

A simple method to extract DNA from hair shafts was developed by using enzymatic laundry powder at the first step of the process. The whole extraction can be finished in less than 2 hours. The simple extraction reagent proposed here contains only two cheap components: ordinary enzymatic laundry powder and PCR buffer. After extraction, an ultra sensitive fluorescent nucleic acid stain, PicoGreen, was used for quantifying trace amount of double-stranded DNA in the solution extracted. For further validation of DNA extraction, four primers were employed to amplify DNA microsatellite loci. Both fluorescence spectroscopy and PCR results suggested that this method can extract DNA from hair shafts with good efficiency and repeatability. The study will greatly facilitate the use of hair shafts in future for DNA analyses on genome-wide scale.


Introduction
Hair shafts as a potential source of DNA is valuable for noninvasive study of human and nonhuman populations. It can be used in genetic analysis to identify individuals and breed in animal husbandry traceability, wild animal germplasm resources protection, forensic medical study and paleontological research, etc. The characteristic of mitochondrial DNA (mtDNA) extracted from hair shafts has already been well described previously [1][2][3][4][5]. In addition, James Robertson et al. [6] described that mtDNA has limited value because mtDNA profiles cannot be compared with national and international databases of Short Tandem Repeat (STR) genotypes. Furthermore, mtDNA cannot discriminate maternal relatives and lack the discriminating power of STR profiles. However, fragmentation of the nuclear DNA (nuDNA) in the hair shafts was considered as a result of the keratinisation process [7] and nuDNA seldom been successfully analyzed. Thus, a major concern in the hair shafts DNA extraction and amplification today is trying to find an efficient, stable and easy way to extract DNA and amplify nuDNA targets.
In the present study, we have developed a simple method to extract DNA from hair shafts. With this approach, it took less than 2 hours to extract DNA and the simple extraction reagent contains only ordinary enzymatic laundry powder and 16 PCR buffer, which was cheap and easily available. Fluorescence spectroscopy approach, Polymerase Chain Reaction (PCR) and on-chipelectrophoresis results confirmed that enzymatic laundry powders could be used to extract DNA and amplify microsatellite markers from hair shafts.

Ethics Statement
The study was approved by the Animal Care and Use Committee of the Institute of Quality Standards and Testing Technology for Agro-products, Chinese Academy of Agricultural Sciences and the owners of the cattle. Permission to collect and use hair shafts samples from cattle in this study were granted by Shandong Kelong Farming Industry Limited Company.

Samples
Hair shafts were collected from cattle (24 pure bred Luxi cattle, in Heze, Shandong Province, China), November 2011. Fresh liver samples of beef cattle were purchased from Beijing Anding Abattoir. The samples were stored at 218uC until DNA extraction.

DNA extraction
Full precautions were taken to prevent external DNA contamination. All experiments were performed following protocols for ancient DNA extraction [8,9]. Laboratories, laboratory ware, plastic ware, reagents, pipettes, pipette tips, benches and equipments were irradiated under ultraviolet light (250 nm for 1 hour) before and after each experiment. In addition, only the ware and pipette tips, which have been steam sterilized (high steam pressure, 121uC for 1 hour) were used in this study. All the steps of the analysis were performed wearing sterile gloves, face masks, hair nets and laboratory coats, under positive pressure in a laminar flow hood. Reagents are molecular grade except enzymatic laundry powder. Negative controls (containing a reagent blank) were included in all assays. Prior to DNA extraction, samples were decontaminated through immersion in 84 TM Disinfectant (the content of effective chlorine is 5-6%) for 30-40 seconds, then rinsed in ddH 2 O three times. The ddH 2 O, which was used to rinse the sample (the third time), was set as a pre-treatment control to ensure there was no contamination. After that, samples were soaked in sterile ethanol, and left to air-dry.
Hair shafts were cut into fragments of about 2 mm. Each sample ( Table 1 and Table 2) was digested in 100 ml of extraction reagent (pH 10.3) for 1.5 hours at 50uC. The extraction reagent contained 3 mg enzymatic laundry powder (Diao TM enzymatic laundry powder made by Nice Group, Keon TM enzymatic laundry powder made by Nafine, and OMO TM enzymatic laundry powder made by Unilever), and 16PCR buffer (20 mM Tris-HCl (pH 8.4), 20 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 1.5 mM MgCl 2 , Tiangen Biotech (Beijing) Co., Ltd.). Besides that, liver DNA was separately extracted in another room using Genomic DNA Purification Kit (Fermentas Life Sciences, Thermo Fisher) as a positive control.
After extraction, extraction solutions were gradually heated up to 95uC to improve extract efficiency [10], and then subject to 95uC for 10 minutes in order to inactivate enzymes in the extraction reagent. The final DNA extracts were stored at 218uC until use.

DNA quantitation
Quant-iT TM PicoGreenH dsDNA reagent (Invitrogen) is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in solution. It is used to help quantify DNA in extraction solution by fluorescence spectroscopy approach. This method is at least 400 times more sensitive than the Hoechst dye based assay [11].
The quantification assay was carried out as described in Chinese Pharmacopoeia (Appendix B, Vol. III) [12] which states that the lower limit of detection of this approach is 0.3 ng/ml and linearity is reported between 1.25-80 ng/ml. Therefore according to the results of published literature [6,13,14], the DNA in hair shafts could be detected by the quantitative assay.  (Table 1). For real-time PCR, it was carried out in 20 ml reaction volumes with the final concentration of 16 Power SYBR Green PCR Master Mix (ABI), 200 nM each primer, and different volumes of template (Table 2). A joint committee of Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics (ISAG) has recommended a list of microsatellite markers for cattle [15]. For the amplification, four markers (CSRM60, 79-115 base pairs (bp); INRA035, 100-124 bp; ETH225, 131-159 bp; and HAUT27, 120-158 bp) were selected from the list and the primers were synthesized by Shanghai SANGON Biological Engineering Technology Services Limited.   The extract solution was used directly as PCR template in the first round. The second round used the product of the first round as its template. The two-round cycling steps were identical and as follows: initial denaturation at 95uC for 10 minutes, followed by 35 cycles at 95uC for 20 seconds, annealing (40 seconds, annealing temperatures are shown in Table 3), extension at 72uC for 1 minute, and a final extension at 72uC for 10 minutes.

Microsatellite amplification
Positive control was amplified in another room, with same condition and same template volume but only the first round PCR was performed.
Then, amplification products (locus CSRM60 and INRA035) were pipette onto DNA Chips (on-chip-electrophoresis, Agilent DNA 1000 Kit, for use with the Agilent 2100 bioanalyzer) and operated as its Guide described, while the others were visualized under UV light on 1.2% agarose gels by staining with GeneGreen (Tiangen Biotech (Beijing) Co., Ltd.).

Statistical Analysis
All statistical analyses were performed with IBM SPSS Statistics (Version 19) software. Calculations were done in Microsoft EXCEL spread sheets. The arithmetic means and standard deviations were used to carry out a standard t-test, and the significance level was measured at less than 0.05. Moreover, Spearman's rho test is used for detecting correlation between the C T values and input DNA amounts.

Results
DNA was extracted from hair shafts of 24 pure bred Luxi cattle. The DNA content of the extract (Table 1) is 8.8760.50 ng/ml (n = 24) and the values of negative control groups are below the limit of detection. The samples were randomly divided into two groups to amplify locus CSRM60 and INRA035 respectively. 11 of the 12 samples were amplified CSRM60 successfully, and 9 of the other 12 samples were amplified INRA035 successfully. No blanks exhibited evidence of contamination. Reagent blank (extraction solution without hair shafts), negative control (ddH 2 O as template), and pre-treatment control (the third time washing water of hair shafts after disinfectant treatment) confirmed the  absence of any external DNA contamination by the results of fluorescence spectroscopy and on-chip-electrophoresis identification tests. On-chip-electrophoresis results were shown in Figure 1. The bands of chip-electrophoresis of hair shaft DNA amplification results were compared with that of liver source DNA in Figure 2 and the results demonstrated the accuracy of our target locus according to the ISAG-FAO document [15].
Moreover, to investigate the best template volume and adequate sample amount, six template volume groups and six sample weight groups were set to amplify locus ETH225 and HAUT27 respectively. In this part, we extracted 144 samples (Table 2), and as shown in Table 4, all of them can yield DNA by using the proposed extraction method with enzymatic laundry powder. The results of real-time PCR (Figure 3, Figure 4 and Table 5) demonstrated that low template amount groups were amplified more efficiently. Aside from the template amount in the amplification, for this approach, sample amount is not a major factor.

Discussion
The fact that enzymatic laundry powder contains protease, lipase, cellulose, surfactant and suspending agent, suggests that it could be used as a good extraction reagent to extract DNA [16] from the hair shafts. In the study, all three brands of enzymatic laundry powder contain anionic surfactant (sodium dodecyl benzene sulfonate), nonionic surfactant (polyoxyethylene ethers), suspending agent, and complex enzyme (including protease), that endow them with the extraction capability. Compared to the other methods (Table 6), it was not only simple and time-efficient, but also showed to be the most cost-effective method.
NuDNA in hair shafts is known for its small quantity [17], and there are large quantities of impurities like keratin and pigments ( Figure 5) which can affect the detection of nucleic acids and seriously inhibit PCR amplification. Ovoid pigment granules are located in the cortex (inner layer) and medulla (core), but the exact location of recoverable nuDNA in the hair shafts remains unclear. According to the literature [14,[18][19][20], we supposed that it is localized in the cuticle (outer layer). For this reason, we tried enzymatic laundry powder with the assumption that it can get enough nuDNA from cuticle as PCR template but less amplification inhibition substances.
As Nozawa and Uchihi et al. [18,21] noted, the impurities like pigments can essentially give the same spectrum between 500 and 230 nm. Therefore, the spectrum of the DNA extract always appears to contain pigments in addition to DNA and some other  . Negative correlation between PCR efficiency and the amount of input template. The data of all three enzymatic laundry powder groups (each detergent group has six sample amount groups: 5 mg, 2 mg, 1 mg, 0.5 mg, 0.2 mg, 0.1 mg; and each sample amount group has six template volume groups: 5 ml, 2 ml, 1 ml, 0.5 ml, 0.2 ml, 0.1 ml) were adopted (target locus ETH225, second round), Spearman's rho = 0.810, P,0.001, n = 107 (one sample has no C T value and has been excluded by the SPSS). 90 of 107 points were in circular ROI box (yellow oval area), mean C T value is 8.7, SD = 4.1; and median C T value is 6.0. doi:10.1371/journal.pone.0069588.g004 substrates which have absorbance at wavelengths lower than 230 nm. It was challenging to quantify DNA amount in extraction solution directly before or without PCR except if the extract has been purified strictly. Another limitation was that sometimes the quantity of hair shafts or samples used for extracting DNA is not enough and the extracted DNA cannot reach the level of detectability (2 ng/ml) used by most detectors. Lack of DNA quantity in the extract leads to more failures in next PCR or other downstream work. Therefore, for the first time, we attempted fluorescence spectroscopy approach in this field and used Quant-iT TM PicoGreenH dsDNA reagent, an ultra-sensitive fluorescent nucleic acid stain to quantify dsDNA in extraction solution.
In this study, it has been proved that the sensitive fluorescent assay can be used to detect the DNA in hair shafts extraction solution directly, and enzymatic laundry powder can be used to extract DNA from the hair shafts. From the results obtained by   HAUT27). Furthermore, the following PCR with high success rate suggested that the low template amount could increase PCR efficiency ( Figure 3, Figure 4 and Table 5). Actually, there is a negative correlation between PCR efficiency and input template amount and small amount of input DNA can decrease the C T values ( Figure 4, Spearman's rho = 0.810, P,0.001, n = 107). The results indicated that there were potential PCR inhibitors in the extraction solution which are the critical factors for successful PCR in our strategy. It is for that very reason that we can amplify targets successfully by using trace input DNA as low as 0.16 pg, and this also made it possible for most of enzymatic detergents to use in our method, although their extraction efficiency has significant differences ( Table 4 and Table 5). According to the literature [18,[21][22][23], we considered that as a compromise between PCR template and amplification inhibition factors. The extraction and two rounds amplification strategy presented above has been performed hundreds of times, but there still a few target loci failed to be amplified, we thought that it may be not caused by unpurified PCR template or non-optimized amplification condition, since it was clear that most of target genes can be amplified successfully. It might be caused by the keratinisation process for its cytolysis [24][25][26]. If the template was cracked, an amplification failure would occur as we described. Therefore, the proposed method using low amount DNA to overcome impurities inhibition on PCR may not of universal appeal e.g. in applications such as Next Generation Sequencing where maintaining overall molecular diversity is essential.
In conclusion, we developed a simple and cost-effective method to extract DNA from hair shafts using enzymatic laundry powder and PCR buffer with high efficiency and even can extract DNA from trace amount (0.1 mg) of hair shafts. The extract could be directly used as template to amplify the target gene. Repeated experiments also showed the good stability and reliability of the approach. The results indicated the simple method has a good practical future regarding its cost-effectiveness and easily operation. Considering hair shafts characteristics of easy to get, transport and store, we believe the proposed method in this study will find more applications in the animal husbandry traceability research, breeding cultivation and wild animal biodiversity study, etc.