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Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples
Forensic Science International: Genetics 6 (2012) 306–309
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples Hongmei Xu 1, Yun Zhao 1, Zhiping Liu, Wei Zhu, Yueqin Zhou, Ziqin Zhao * Department of Forensic Medicine, Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2010 Received in revised form 16 April 2011 Accepted 13 June 2011
DNA methylation is an important event in epigenetic changes in cells, and a fundamental regulator of gene transcription. Bisulfite genomic sequencing is a powerful technique used in studies of DNA methylation. However, the established procedures often require relatively large amounts of DNA. In everyday practice, samples submitted for analysis might contain very small amounts of poor quality material, as is often the case with forensic stain samples. In this study, we assess a modified, more efficient method of bisulfite genomic sequencing. Genomic DNA extracted from 3-mm dried blood spots using QIAamp micro kit was treated with sodium bisulfite (using EpiTect kit). Subsequent methylationspecific PCR (MSP) followed by DNA sequencing displayed the differentially methylated region of imprinted gene SNRPN. Our results show that this new combination of efficient DNA extraction and bisulfite treatment provides high quality conversion of unmethylated cytosine to uracil for bisulfite genomic sequencing analysis. This reliable method substantially improves the DNA methylation analysis of forensic stain samples. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic biology DNA methylation Bisulfite genomic sequencing Dried blood spot microvolume sample MSP
1. Introduction DNA methylation, which often occurs at 50 -CpG-30 sites, is considered a significant factor in epigenetic regulation of gene activity [1]. A large body of literature has demonstrated that abnormal methylation patterns could result in cancer, imprinting disorders or other diseases [2–6]. Differential methylation patterns have been associated with schizophrenia and bipolar disorder [7], alcohol dependence [8], and suicide [9]. Elucidation of aberrant DNA methylation patterns would be of great benefit to our understanding of these phenomena; one of the best approaches in these types of study is the bisulfite genomic sequencing method. After bisulfite treatment of DNA, unmethylated cytosine converts to uracil, whereas methylation of cytosine blocks this reaction [10]. After PCR amplification, the uracil residues appear as thymines. However, conventional bisulfite sequencing procedure demands substantial quantities of DNA to compensate for degradation and loss during bisulfite conversion. DNA microvolume samples isolated from dried blood spots, often used in forensics, are not the ideal starting material for methylation analysis. They contain small amounts of DNA, usually of rather poor quality. In most cases, they do not withstand well the harsh conditions of bisulfite conversion process.
* Corresponding author. Tel.: +86 21 54237668; fax: +86 21 54237668. E-mail addresses: [email protected], [email protected] (Z. Zhao). 1 These authors contributed equally to this paper. 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.06.007
Recently, dried blood spots have become preferred forensic samples in many forensic laboratories worldwide, as they are easy to collect, transport and store. Since 2008, dried blood spots have constituted about 90% of the evidence samples received in our laboratory. However, prolonged storage of dried blood spots can lead to DNA fragmentation [11]. Methods for bisulfite genomic sequencing from blood spots were first reported by Wong et al. [12]. They described a combination of genomic DNA extraction using the ChargeSwitch Forensic DNA Purification kit and MethyIEasy bisulfite modification, used with DNA from archived Guthrie blood spots. Here, we use a bisulfite conversion protocol recently developed by Qiagen for genomic DNA isolated from dried blood spot microvolume samples. After bisulfite treatment, we examined the differentially methylated region (DMR) of maternal imprinted gene SNRPN using MSP and direct sequencing. Our results demonstrate that this new bisulfite genomic sequencing procedure, adapted to the type of forensic samples described above, can be successfully applied in DNA methylation analysis.
2. Materials and methods 2.1. Samples Blood samples from 100 individuals were collected by the members of the Forensic Biology Laboratory, Department of Forensic Medicine, Shanghai Medical College, Fudan University. All donors signed the appropriate informed consent forms. 10 mL of blood was spotted on ordinary filter paper, dried, and stored at
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
307
room temperature for one year. All the procedures were approved by the Ethics Committee of Fudan University. 2.2. DNA isolation Genomic DNA was extracted from 3-mm diameter punches of dried blood spots using QIAamp DNA Micro kit (Qiagen, Germany), in a final volume of 20 mL. It was separated on 1% agarose gel and stained with ethidium bromide. The yield was determined in NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). Genomic DNA was isolated in a total volume of 20 mL with a concentration of a range of 5.34–25.11 ng/mL. The total DNA yield was 100–500 ng. 2.3. Bisulfite conversion 50–300 ng of genomic DNA in a volume of 10 mL was converted using EpiTect Bisulfite kit. Bisulfite conversion was performed in 10 mL of RNase-free water, 85 mL of Bisulfite Mix solution, and 35 mL of DNA Protect Buffer. The total reaction volume was 140 mL. We performed the procedure according to the manufacturer’s instructions. 2.4. Methylation-specific polymerase chain reaction (MSP) Primers [13] targeted to SNRPN DMR gene were as follows: methylated pair (M-up: 50 -taaataagtacgtttgcgcggtc-30 ; M-down: 50 -aaccttacccgctccatcgcg-30 ) and unmethylated pair (U-up: 50 tgaggttggtgtgtatgtttaggt-30 ; U-down: 50 -acatcaaacatctccaacaacca30 ). MSP reactions were performed in ABI 9700 PCR system in a total volume of 20 mL, using the mixture containing 2 Taq PCR MasterMix (0.1 U Taq Polymerase/mL, 500 mM dNTP, 20 mM Tris– HCl, 100 mM KCl, 3 mM MgCl2, TIANGEN BIOTECH, Shanghai, China), 1 mM primers, and bisulfite modified DNA (10 ng). We used the following amplification conditions: 958 for 15 min, followed by 35 cycles at 948 for 30 s, at 628 for 30 s, and 728 for 30 s. After MSP, a 2.5% agarose gel and staining with ethidium bromide was used to identify the products. Positive control DNA samples (CpG Genome Universal Methylated, Chemicon International) were used in all MSP reactions. 2.5. DNA sequencing The PCR products were extracted from the 2.5% agarose gel stained with ethidium bromide. Purified DNA samples were sent to Invitrogen Co. (Shanghai, China) for sequencing on ABI 3730. 3. Results We used 1% agarose gel electrophoresis and ethidium bromide staining to show the genomic DNA extracted from 3-mm diameter punches of dried blood spots. The size of DNA was 10 kb (Fig. 1). NanoDrop data showed the yield and quality of extracted DNA using QIAamp DNA Micro kit. DNA yield was 100–500 ng. Absorption 260/ 280 ratios of 100 samples were in a range from 1.40 to 1.90 (Table 1). The original samples chosen for bisulfite conversion contained 5.34– 25.11 ng/mL DNA in a volume of 10 mL (Fig. 2). MSP products were identified on a 2.5% agarose gel stained with ethidium bromide. A 174 bp and a 100 bp band correspond to the methylated maternal allele and unmethylated paternal allele, respectively. In Fig. 3, the maternal and paternal alleles in two individuals (samples 1 and 2) are visible. Another individual (sample 3) displays a clear 174 bp band but an extremely faint 100 bp band represents for the paternal allele. The 100 bp band in some samples, however, is invisible (data not shown). The failed detection of unmethylated paternal allele may due to some
Fig. 1. 1% agarose gel stained with ethidium bromide used for an assessment of the integrity of genomic DNA extracted from dried blood spots. DNA was 10 kb in length, of good integrity. DNA markers from bottom to top (kb): 1, 2, 3, 4, 5, 6, 7, 8, 9 (Takara, Japan).
Table 1 NanoDrop data for 100 input DNA samples. A260/280
Number
DNA yield (ng)
1.40–1.60 1.61–1.80 1.81–1.90
17 49 34
100–150 107–500 200–500
potential relationships with diseases. For example, the 100 bp band cannot be observed in some patients with acute myeloid leukemia, myelodysplastic syndromes, central neurocytomas, and pediatric germ cell tumors [13–15]. The 174 bp band corresponding to the methylated maternal allele can be amplified completely in all of 100 samples underwent MSP. We then compared the original sequences of SNRPN DMR gene with the results of direct sequencing. In order to guarantee the substantial amount of PCR products required for sequencing, typical DNA samples with a bright 174 bp band were picked out according to 2.5% agarose gel images. Fig. 4 showed one of the sequencing results, which confirmed that EpiTect bisulfite procedure can offer complete conversion of unmethylated cytosine to uracil in bisulfite genomic sequencing analysis on dried blood spot microvolume samples (Fig. 4). Similar sequencing results were found for other typical samples. 4. Discussion DNA methylation plays a major role in both normal and pathological processes such as epigenetic reprogramming [16] and
Fig. 2. Input DNA in each of 100 samples: volume of 10 mL, concentration 5.34– 25.11 ng/mL. The concentration variation: 46 samples from 5.00 to 10.00 ng/mL; 27 samples from 10.01 to 15.00 ng/mL; 9 samples from 15.01 to 20.00 ng/mL; 18 samples in the range from 20.01 to 26.00 ng/mL.
308
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
Fig. 3. MSP of SNRPN DMR gene. Primer sets for MSP were either methylated (M) or unmethylated (U). Negative control (no DNA) was pure water. Positive control, the universally methylated DNA, shows positive for M only. In samples 1 and 2, we detected both a 174 bp PCR product of the methylated maternal allele and a 100 bp band of unmethylated paternal allele. Sample 3 shows only methylated band. DNA marker sizes from bottom to top (bp): 100, 120, 140, 160, 180, 200, 300, 400, 500 (Takara, Japan).
Fig. 4. Comparison between the primary sequence of SNRPN DMR gene and the bisulfite modified sequence. After MSP, unmethylated cytosine was transformed to thymine and left 5-methyl-C unchanged.
cancer [17]. Many current forensic biology studies focus on the improvements in the techniques allowing better understanding of the DNA methylation patterns. Forensic samples often contain only a few cells in the form of contact stains from handled objects, surfaces or clothes [18]. The most common among these stain-samples are dried blood stains. Most stains found at crime scenes are of that type, and they can be collected on filter paper, cotton swabs, or moist piece of cotton. Although dried blood stain-samples are easy to handle and store, standard extraction procedures give DNA with low yield and poor quality. The combination of ChargeSwitch Forensic DNA Purification kit and MethyIEasy bisulfite modification is the only prior report of bisulfite sequencing from Archived Guthrie blood spots. The ChargeSwitch Forensic DNA Purification kit allows DNA yield of 45–100 ng per sample. In our study, total yield for DNA isolated from 3-mm dried blood spots was 100–500 ng per sample. The kit we used, QIAamp DNA Micro kit, has been specifically designed for DNA extraction from microvolume blood samples or dried blood spots. In this study, 10 mL of blood was spotted on the ordinary filter paper, dried, and stored at room temperature for a year. The QIAamp DNA Micro kit for extracting DNA from dried blood spots resulted in higher yields. More particularly, its unique silica membrane column preserves DNA integrity during the process of
extraction and helps to remove the contaminants. DNA size in all of 100 samples was below 10 kb but no significant fragmentation was observed. However, the concentration of genomic DNA extracted in our study was still low: 5.34–25.11 ng/mL. In the past, this would have limited their use in the subsequent bisulfite genomic sequencing procedure. Before Wong et al. [12], bisulfite genomic sequencing with limited amounts of DNA from aged samples other than blood has been developed by Millar et al. [19]. Unfortunately, the amounts of DNA before bisulfite modification failed to be found in Millar’s method. The procedure of EpiTect Bisulfite kit, unlike the conditions employed in conventional bisulfite genomic sequencing methods, allows a starting DNA amount of less than 1 ng due to its unique DNA protection buffer which can help maximizing DNA survival in the high bisulfite salt concentrations, high temperature and low pH conditions, preventing material loss and fragmentation. Furthermore, carrier RNA provided in EpiTect kit promotes the bisulfite modification of DNA when starting amounts are less than 100 ng. We assessed the feasibility of this kit to perform bisulfite modification on dried blood spot microvolume samples. The detection for the methylation pattern of SNRPN DMR gene using MSP follow by 2.5% agarose gel electrophoresis supported the notion that genomic DNA extracted from dried blood spot
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
microvolume samples can be completely modified by its efficient protocol. DNA sequencing also demonstrated the high quality conversion of unmethylated cytosine to uracil during our bisulfite modification procedure. To our knowledge, this is the first report on performing QIAamp DNA micro kit on dried blood spot microvolume samples followed by EpiTect bisulfite kit. We conclude that this particular combination of DNA extraction and bisulfite conversion results in successful bisulfite conversion when working with DNA isolated from dried blood spot microvolume samples. Genomic DNA isolated from such samples is of high purity and good integrity. Modified DNA can be amplified by MSP and regions of interest sequenced to perform methylation analysis. The procedure is easy to introduce in a forensic biology laboratory and gives reproducible and reliable results. This new method extends the scope of the DNA methylation analysis of forensic stain samples and provides a new tool for methylation pattern detection in the genes of potential forensic interest. Conflict of interest None. References [1] H. Hayatsu, The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research, Mutat. Res. 659 (2008) 77–82. [2] K. Hibi, T. Goto, H. Mizukami, Y. Kitamura, M. Sakata, M. Saito, K. Ishibashi, G. Kigawa, H. Nemoto, Y. Sanada, MGMT gene is aberrantly methylated from the early stages of colorectal cancers, Hepatogastroenterology 56 (2009) 1642–1644. [3] Z. Herceg, T. Ushijima, Introduction: epigenetics and cancer, Adv. Genet. 70 (2010) 1–23. [4] H. Judson, B.E. Hayward, E. Sheridan, D.T. Bonthron, A global disorder of imprinting in the human female germ line, Nature 416 (2002) 539–542. [5] M. Procter, L.S. Chou, W. Tang, M. Jama, R. Mao, Molecular diagnosis of ParderWilli and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification, Clin. Chem. 52 (2006) 1276–1283.
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[6] J. Liu, J.G. Nealon, L.S. Weinstein, Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB, Hum. Mol. Genet. 14 (2005) 95–102. [7] H.M. Abdolmaleky, K.H. Cheng, S.V. Faraone, M. Wilcox, S.J. Glatt, F. Gao, C.L. Smith, R. Shafa, B. Aeali, J. Carnevale, H. Pan, P. Papageorgis, J.F. Ponte, V. Sivaraman, M.T. Tsuang, S. Thiagalingam, Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder, Hum. Mol. Genet. 15 (2006) 3132–3145. [8] M.A. Muschler, T. Hillemacher, C. Kraus, J. Kornhuber, S. Bleich, H. Frieling, DNA methylation of the POMC gene promoter is associated with craving in alcohol dependence, J. Neural Transm. 117 (2010) 513–519. [9] M.O. Poulter, L. Du, I.C.G. Weaver, M. Palkovits, G. Faludi, Z. Merali, M. Szyf, H. Anisman, GABAA receptor promoter hypermethylation in suicide brain: implications for the involvement of epigenetic processes, Biol. Psychiatr. 64 (2008) 645–652. [10] J. Lewin, A.O. Schmitt, P. Adorja´n, T. Hildmann, C. Piepenbrock, Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates, Bioinformatics 20 (2004) 3005–3012. [11] G.S. Makowski, E.L. Davis, S.M. Hopfer, The effect of storage on Guthrie cards: implications for deoxyribonucleic acid amplification, Ann. Clin. Lab. Sci. 26 (1996) 458–469. [12] N.C. Wong, R. Morley, R. Saffery, J. Craig, Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis, Biotechniques 45 (2008) 423– 424, 426, 428, 430. [13] L. Benetatos, E. Hatzimichael, A. Dasoula, G. Dranitsaris, S. Tsiara, M. Syrrou, I. Georgiou, K.L. Bourantas, CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes, Leukemia Res. 34 (2010) 148–153. [14] A. Korshunov, R. Sycheva, A. Golanov, Recurrent cytogenetic aberrations in central neurocytomas and their biological relevance, Acta Neuropathol. 113 (2007) 303–312. [15] D.T. Schneider, A.E. Schuster, M.K. Fritsch, J. Hu, T. Olson, S. Lauer, U. Go¨bel, E.J. Perlman, Multipoint imprinting analysis indicates a common precursor cell for gonadal and nongonadal pediatric germ cell tumors, Cancer Res. 61 (2001) 7268–7276. [16] F.F. Costa, E.A. Seftor, J.M. Bischof, D.A. Kischmann, L. Strizzi, K. Arndt, M. de Fatima Bonaldo, M.B. Soares, M.J. Hendrix, Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment, Epigenomics 1 (2009) 387–398. [17] R.J. Preston, Epigenetic processes and cancer risk assessment, Mutat. Res. 616 (2007) 7–10. [18] P. Wiegand, C. Heimbold, R. Klein, U. Immel, D. Stiller, M. Klintschar, Transfer of biological stains from different surfaces, Int. J. Legal Med. (2010) (Epub ahead of print). [19] D.S. Millar, P.M. Warnecke, J.R. Melki, S.J. Clark, Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells, Methods 27 (2002) 108–113.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples Hongmei Xu 1, Yun Zhao 1, Zhiping Liu, Wei Zhu, Yueqin Zhou, Ziqin Zhao * Department of Forensic Medicine, Shanghai Medical College, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2010 Received in revised form 16 April 2011 Accepted 13 June 2011
DNA methylation is an important event in epigenetic changes in cells, and a fundamental regulator of gene transcription. Bisulfite genomic sequencing is a powerful technique used in studies of DNA methylation. However, the established procedures often require relatively large amounts of DNA. In everyday practice, samples submitted for analysis might contain very small amounts of poor quality material, as is often the case with forensic stain samples. In this study, we assess a modified, more efficient method of bisulfite genomic sequencing. Genomic DNA extracted from 3-mm dried blood spots using QIAamp micro kit was treated with sodium bisulfite (using EpiTect kit). Subsequent methylationspecific PCR (MSP) followed by DNA sequencing displayed the differentially methylated region of imprinted gene SNRPN. Our results show that this new combination of efficient DNA extraction and bisulfite treatment provides high quality conversion of unmethylated cytosine to uracil for bisulfite genomic sequencing analysis. This reliable method substantially improves the DNA methylation analysis of forensic stain samples. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic biology DNA methylation Bisulfite genomic sequencing Dried blood spot microvolume sample MSP
1. Introduction DNA methylation, which often occurs at 50 -CpG-30 sites, is considered a significant factor in epigenetic regulation of gene activity [1]. A large body of literature has demonstrated that abnormal methylation patterns could result in cancer, imprinting disorders or other diseases [2–6]. Differential methylation patterns have been associated with schizophrenia and bipolar disorder [7], alcohol dependence [8], and suicide [9]. Elucidation of aberrant DNA methylation patterns would be of great benefit to our understanding of these phenomena; one of the best approaches in these types of study is the bisulfite genomic sequencing method. After bisulfite treatment of DNA, unmethylated cytosine converts to uracil, whereas methylation of cytosine blocks this reaction [10]. After PCR amplification, the uracil residues appear as thymines. However, conventional bisulfite sequencing procedure demands substantial quantities of DNA to compensate for degradation and loss during bisulfite conversion. DNA microvolume samples isolated from dried blood spots, often used in forensics, are not the ideal starting material for methylation analysis. They contain small amounts of DNA, usually of rather poor quality. In most cases, they do not withstand well the harsh conditions of bisulfite conversion process.
* Corresponding author. Tel.: +86 21 54237668; fax: +86 21 54237668. E-mail addresses: [email protected], [email protected] (Z. Zhao). 1 These authors contributed equally to this paper. 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.06.007
Recently, dried blood spots have become preferred forensic samples in many forensic laboratories worldwide, as they are easy to collect, transport and store. Since 2008, dried blood spots have constituted about 90% of the evidence samples received in our laboratory. However, prolonged storage of dried blood spots can lead to DNA fragmentation [11]. Methods for bisulfite genomic sequencing from blood spots were first reported by Wong et al. [12]. They described a combination of genomic DNA extraction using the ChargeSwitch Forensic DNA Purification kit and MethyIEasy bisulfite modification, used with DNA from archived Guthrie blood spots. Here, we use a bisulfite conversion protocol recently developed by Qiagen for genomic DNA isolated from dried blood spot microvolume samples. After bisulfite treatment, we examined the differentially methylated region (DMR) of maternal imprinted gene SNRPN using MSP and direct sequencing. Our results demonstrate that this new bisulfite genomic sequencing procedure, adapted to the type of forensic samples described above, can be successfully applied in DNA methylation analysis.
2. Materials and methods 2.1. Samples Blood samples from 100 individuals were collected by the members of the Forensic Biology Laboratory, Department of Forensic Medicine, Shanghai Medical College, Fudan University. All donors signed the appropriate informed consent forms. 10 mL of blood was spotted on ordinary filter paper, dried, and stored at
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
307
room temperature for one year. All the procedures were approved by the Ethics Committee of Fudan University. 2.2. DNA isolation Genomic DNA was extracted from 3-mm diameter punches of dried blood spots using QIAamp DNA Micro kit (Qiagen, Germany), in a final volume of 20 mL. It was separated on 1% agarose gel and stained with ethidium bromide. The yield was determined in NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). Genomic DNA was isolated in a total volume of 20 mL with a concentration of a range of 5.34–25.11 ng/mL. The total DNA yield was 100–500 ng. 2.3. Bisulfite conversion 50–300 ng of genomic DNA in a volume of 10 mL was converted using EpiTect Bisulfite kit. Bisulfite conversion was performed in 10 mL of RNase-free water, 85 mL of Bisulfite Mix solution, and 35 mL of DNA Protect Buffer. The total reaction volume was 140 mL. We performed the procedure according to the manufacturer’s instructions. 2.4. Methylation-specific polymerase chain reaction (MSP) Primers [13] targeted to SNRPN DMR gene were as follows: methylated pair (M-up: 50 -taaataagtacgtttgcgcggtc-30 ; M-down: 50 -aaccttacccgctccatcgcg-30 ) and unmethylated pair (U-up: 50 tgaggttggtgtgtatgtttaggt-30 ; U-down: 50 -acatcaaacatctccaacaacca30 ). MSP reactions were performed in ABI 9700 PCR system in a total volume of 20 mL, using the mixture containing 2 Taq PCR MasterMix (0.1 U Taq Polymerase/mL, 500 mM dNTP, 20 mM Tris– HCl, 100 mM KCl, 3 mM MgCl2, TIANGEN BIOTECH, Shanghai, China), 1 mM primers, and bisulfite modified DNA (10 ng). We used the following amplification conditions: 958 for 15 min, followed by 35 cycles at 948 for 30 s, at 628 for 30 s, and 728 for 30 s. After MSP, a 2.5% agarose gel and staining with ethidium bromide was used to identify the products. Positive control DNA samples (CpG Genome Universal Methylated, Chemicon International) were used in all MSP reactions. 2.5. DNA sequencing The PCR products were extracted from the 2.5% agarose gel stained with ethidium bromide. Purified DNA samples were sent to Invitrogen Co. (Shanghai, China) for sequencing on ABI 3730. 3. Results We used 1% agarose gel electrophoresis and ethidium bromide staining to show the genomic DNA extracted from 3-mm diameter punches of dried blood spots. The size of DNA was 10 kb (Fig. 1). NanoDrop data showed the yield and quality of extracted DNA using QIAamp DNA Micro kit. DNA yield was 100–500 ng. Absorption 260/ 280 ratios of 100 samples were in a range from 1.40 to 1.90 (Table 1). The original samples chosen for bisulfite conversion contained 5.34– 25.11 ng/mL DNA in a volume of 10 mL (Fig. 2). MSP products were identified on a 2.5% agarose gel stained with ethidium bromide. A 174 bp and a 100 bp band correspond to the methylated maternal allele and unmethylated paternal allele, respectively. In Fig. 3, the maternal and paternal alleles in two individuals (samples 1 and 2) are visible. Another individual (sample 3) displays a clear 174 bp band but an extremely faint 100 bp band represents for the paternal allele. The 100 bp band in some samples, however, is invisible (data not shown). The failed detection of unmethylated paternal allele may due to some
Fig. 1. 1% agarose gel stained with ethidium bromide used for an assessment of the integrity of genomic DNA extracted from dried blood spots. DNA was 10 kb in length, of good integrity. DNA markers from bottom to top (kb): 1, 2, 3, 4, 5, 6, 7, 8, 9 (Takara, Japan).
Table 1 NanoDrop data for 100 input DNA samples. A260/280
Number
DNA yield (ng)
1.40–1.60 1.61–1.80 1.81–1.90
17 49 34
100–150 107–500 200–500
potential relationships with diseases. For example, the 100 bp band cannot be observed in some patients with acute myeloid leukemia, myelodysplastic syndromes, central neurocytomas, and pediatric germ cell tumors [13–15]. The 174 bp band corresponding to the methylated maternal allele can be amplified completely in all of 100 samples underwent MSP. We then compared the original sequences of SNRPN DMR gene with the results of direct sequencing. In order to guarantee the substantial amount of PCR products required for sequencing, typical DNA samples with a bright 174 bp band were picked out according to 2.5% agarose gel images. Fig. 4 showed one of the sequencing results, which confirmed that EpiTect bisulfite procedure can offer complete conversion of unmethylated cytosine to uracil in bisulfite genomic sequencing analysis on dried blood spot microvolume samples (Fig. 4). Similar sequencing results were found for other typical samples. 4. Discussion DNA methylation plays a major role in both normal and pathological processes such as epigenetic reprogramming [16] and
Fig. 2. Input DNA in each of 100 samples: volume of 10 mL, concentration 5.34– 25.11 ng/mL. The concentration variation: 46 samples from 5.00 to 10.00 ng/mL; 27 samples from 10.01 to 15.00 ng/mL; 9 samples from 15.01 to 20.00 ng/mL; 18 samples in the range from 20.01 to 26.00 ng/mL.
308
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
Fig. 3. MSP of SNRPN DMR gene. Primer sets for MSP were either methylated (M) or unmethylated (U). Negative control (no DNA) was pure water. Positive control, the universally methylated DNA, shows positive for M only. In samples 1 and 2, we detected both a 174 bp PCR product of the methylated maternal allele and a 100 bp band of unmethylated paternal allele. Sample 3 shows only methylated band. DNA marker sizes from bottom to top (bp): 100, 120, 140, 160, 180, 200, 300, 400, 500 (Takara, Japan).
Fig. 4. Comparison between the primary sequence of SNRPN DMR gene and the bisulfite modified sequence. After MSP, unmethylated cytosine was transformed to thymine and left 5-methyl-C unchanged.
cancer [17]. Many current forensic biology studies focus on the improvements in the techniques allowing better understanding of the DNA methylation patterns. Forensic samples often contain only a few cells in the form of contact stains from handled objects, surfaces or clothes [18]. The most common among these stain-samples are dried blood stains. Most stains found at crime scenes are of that type, and they can be collected on filter paper, cotton swabs, or moist piece of cotton. Although dried blood stain-samples are easy to handle and store, standard extraction procedures give DNA with low yield and poor quality. The combination of ChargeSwitch Forensic DNA Purification kit and MethyIEasy bisulfite modification is the only prior report of bisulfite sequencing from Archived Guthrie blood spots. The ChargeSwitch Forensic DNA Purification kit allows DNA yield of 45–100 ng per sample. In our study, total yield for DNA isolated from 3-mm dried blood spots was 100–500 ng per sample. The kit we used, QIAamp DNA Micro kit, has been specifically designed for DNA extraction from microvolume blood samples or dried blood spots. In this study, 10 mL of blood was spotted on the ordinary filter paper, dried, and stored at room temperature for a year. The QIAamp DNA Micro kit for extracting DNA from dried blood spots resulted in higher yields. More particularly, its unique silica membrane column preserves DNA integrity during the process of
extraction and helps to remove the contaminants. DNA size in all of 100 samples was below 10 kb but no significant fragmentation was observed. However, the concentration of genomic DNA extracted in our study was still low: 5.34–25.11 ng/mL. In the past, this would have limited their use in the subsequent bisulfite genomic sequencing procedure. Before Wong et al. [12], bisulfite genomic sequencing with limited amounts of DNA from aged samples other than blood has been developed by Millar et al. [19]. Unfortunately, the amounts of DNA before bisulfite modification failed to be found in Millar’s method. The procedure of EpiTect Bisulfite kit, unlike the conditions employed in conventional bisulfite genomic sequencing methods, allows a starting DNA amount of less than 1 ng due to its unique DNA protection buffer which can help maximizing DNA survival in the high bisulfite salt concentrations, high temperature and low pH conditions, preventing material loss and fragmentation. Furthermore, carrier RNA provided in EpiTect kit promotes the bisulfite modification of DNA when starting amounts are less than 100 ng. We assessed the feasibility of this kit to perform bisulfite modification on dried blood spot microvolume samples. The detection for the methylation pattern of SNRPN DMR gene using MSP follow by 2.5% agarose gel electrophoresis supported the notion that genomic DNA extracted from dried blood spot
H. Xu et al. / Forensic Science International: Genetics 6 (2012) 306–309
microvolume samples can be completely modified by its efficient protocol. DNA sequencing also demonstrated the high quality conversion of unmethylated cytosine to uracil during our bisulfite modification procedure. To our knowledge, this is the first report on performing QIAamp DNA micro kit on dried blood spot microvolume samples followed by EpiTect bisulfite kit. We conclude that this particular combination of DNA extraction and bisulfite conversion results in successful bisulfite conversion when working with DNA isolated from dried blood spot microvolume samples. Genomic DNA isolated from such samples is of high purity and good integrity. Modified DNA can be amplified by MSP and regions of interest sequenced to perform methylation analysis. The procedure is easy to introduce in a forensic biology laboratory and gives reproducible and reliable results. This new method extends the scope of the DNA methylation analysis of forensic stain samples and provides a new tool for methylation pattern detection in the genes of potential forensic interest. Conflict of interest None. References [1] H. Hayatsu, The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research, Mutat. Res. 659 (2008) 77–82. [2] K. Hibi, T. Goto, H. Mizukami, Y. Kitamura, M. Sakata, M. Saito, K. Ishibashi, G. Kigawa, H. Nemoto, Y. Sanada, MGMT gene is aberrantly methylated from the early stages of colorectal cancers, Hepatogastroenterology 56 (2009) 1642–1644. [3] Z. Herceg, T. Ushijima, Introduction: epigenetics and cancer, Adv. Genet. 70 (2010) 1–23. [4] H. Judson, B.E. Hayward, E. Sheridan, D.T. Bonthron, A global disorder of imprinting in the human female germ line, Nature 416 (2002) 539–542. [5] M. Procter, L.S. Chou, W. Tang, M. Jama, R. Mao, Molecular diagnosis of ParderWilli and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification, Clin. Chem. 52 (2006) 1276–1283.
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