Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples.

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 methylation-specific 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.

[1]  R. Preston.  Epigenetic processes and cancer risk assessment. , 2007, Mutation research.

[2]  R. Mao,et al.  Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. , 2006, Clinical chemistry.

[3]  H. Nemoto,et al.  WNT5A gene is aberrantly methylated from the early stages of colorectal cancers. , 2009, Hepato-gastroenterology.

[4]  M. Hendrix,et al.  Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment. , 2009, Epigenomics.

[5]  L. Weinstein,et al.  Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB. , 2005, Human molecular genetics.

[6]  N. Wong,et al.  Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis. , 2008, BioTechniques.

[7]  Jörn Lewin,et al.  Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates , 2004, Bioinform..

[8]  Susan J Clark,et al.  Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells. , 2002, Methods.

[9]  A. Korshunov,et al.  Recurrent cytogenetic aberrations in central neurocytomas and their biological relevance , 2007, Acta Neuropathologica.

[10]  Johannes Kornhuber,et al.  DNA methylation of the POMC gene promoter is associated with craving in alcohol dependence , 2010, Journal of Neural Transmission.

[11]  G. Dranitsaris,et al.  CpG methylation analysis of the MEG3 and SNRPN imprinted genes in acute myeloid leukemia and myelodysplastic syndromes. , 2010, Leukemia research.

[12]  S. Lauer,et al.  Multipoint imprinting analysis indicates a common precursor cell for gonadal and nongonadal pediatric germ cell tumors. , 2001, Cancer research.

[13]  D. Bonthron,et al.  A global disorder of imprinting in the human female germ line , 2002, Nature.

[14]  H. Hayatsu.  The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. , 2008, Mutation research.

[15]  S. Hopfer,et al.  The effect of storage on Guthrie cards: implications for deoxyribonucleic acid amplification. , 1996, Annals of clinical and laboratory science.

[16]  Z. Herceg,et al.  Introduction: epigenetics and cancer. , 2010, Advances in genetics.

[17]  S. Faraone,et al.  Hypomethylation of MB-COMT promoter is a major risk factor for schizophrenia and bipolar disorder. , 2006, Human molecular genetics.

[18]  Miklós Palkovits,et al.  GABAA Receptor Promoter Hypermethylation in Suicide Brain: Implications for the Involvement of Epigenetic Processes , 2008, Biological Psychiatry.

[19]  M. Klintschar,et al.  Transfer of biological stains from different surfaces , 2011, International Journal of Legal Medicine.