title: Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification.
abstract: Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.
authors.affiliation: Department of Forensic Science, Shanxi Medical University, Taiyuan, 030001, China; Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Bai
authors.affiliation: Department of Forensic Science, Shanxi Medical University, Taiyuan, 030001, China; Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Yan
authors.affiliation: Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China; Department of Histology and Embryology, Shanxi Medical University, Taiyuan, 030001, China.
authors.forename: Ximei
authors.initials: X
authors.lastname: Cao
authors.affiliation: Department of Forensic Science, Shanxi Medical University, Taiyuan, 030001, China; Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China.
authors.forename: Linna
authors.initials: L
authors.lastname: Jia
authors.affiliation: Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China.
authors.forename: Ce
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Forensic Science, Shanxi Medical University, Taiyuan, 030001, China; Scientific Research & Experimental Center, Shanxi Medical University, Taiyuan, 30001, China. Electronic address: guo8dawei@aliyun.com.
authors.forename: Dawei
authors.initials: D
authors.lastname: Guo
mesh_terms: D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D018392:Genomic Imprinting; D006801:Humans; D008297:Male; D062085:RNA, Long Noncoding / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D004262:DNA Restriction Enzymes
keywords: H19 imprinting control region; Methylation-sensitive restriction enzyme; Real time PCR; Sperm DNA