| OBJECTIVES In the present study, we will develop a simple and fast methylationvariable position (MVP) analysis method--post fast-methylation-sensitive-restriction-enzyme digestion PCR melting-curve-analysis (PDP-MCA). By which, themethylation status of a group of MVPs will be investigated in forensic stains. Then,the feasibility of using these MVPs as stain markers will be evaluated. The ultimatepurpose is searching for new markers and developing the corresponding detectionmethod for forensic stain identifications. METHODS The development of PDP-MCA: Five groups of MVPs in documented differentially methylated regions (DMR)was selected as candidate markers. For these loci, amplicons with different Tm valuewere designed. By using Fastdigest MSREs and MCA technique, a simple sequentialreaction which integrated multiple reactions (methylation sensitive restriction enzymedigestion, multiplex amplification, melting curve detection) into one tube wasdeveloped. After analyzing the data with Rotor-Gene2.0.2.4software, the MCAprofile for each sample was generated. The methylation level of each MVPs wasdetermined by its amplicon peak height, which was derived from the MCA data inOrigin8software. To validate this method, methylation level of the same samplesdetected by this improved method and the traditional MSRE-PCR was compared.Sample Investigation and marker validation: By PDP-MCA,84stain samples (blood20, saliva20, semen20, vaginal fluid12, menstrual blood12) collected from thehealthy donors were investigated. The MCA/HRM profile generated were comparedand grouped. The normalized fluorescence decreases for each amplicon in all sampleswere collected, for which the discriminant analysis was performed by StatistiXLsoftware. RESULTS The multiplex amplification and MCA analysis of fouramplicons with Tm difference more than2℃were successful. The melt peaks ofeach amplicon separated well. MCA can realize fast and closed-tube detection of PCR products, which are a promising alternative for electrophoresis. With the FastDigestMSREs, three steps of the traditional MSRE-PCR (digestion, amplification anddetection) can be integrated into a single tube. By PDP-MCA, cell-specific MCAprofiles can be generated in four hours. Compared with the control tube, the peakheight and area of the sample tube changed tissue-specifically. The methylation statusreflected by these changes conformed to the results detected by traditional MSRE-PCRand the related reports previously. Under the present conditions, MCA profile can begenerated from0.5ng genomic DNA repeatedly. No significant profile changes werefound for template variations between0.5~20ng.(2) By PDP-MCA, the differentstains investigated ((blood, saliva, semen, vaginal fluid, menstrual blood)) havedifferent MCA profiles. The difference HRM profiles can be clearly grouped into3main categories: male sex stains, female sex stains, and non-sex stains. Bydiscriminant analysis,6linear classification functions were derived from the sampledata: Yblood＝215.889X1＋351.518X2＋184.426X3＋187.220X4-8944.600, Y saliva＝218.104X1＋357.846X2＋185.441X3＋189.631X4-9160.218, Y semen＝211.353X1＋336.697X2＋181.563X3＋182.828X4-8479.036, Y vaginal fluid＝212.469X1＋348.672X2＋180.079X3＋189.103X4-8702.051, Y menstrual blood＝214.026X1＋349.480X2＋182.364X3＋189.059X4-8812.421, Y control＝209.004X1＋337.309X2＋178.322X3＋187.022X4-8367.215. With these functions, the overall correctclassification rate for training samples was97.2%. Except for the blood stains(83.3%), the correct classification rates for other strains were100%. CONLUSIONS(1) PDP-MCA is a simple and fast DNA methylation analysis technique, by whichone-step detection of multiplex MVPs can be realized in a single closed-tube.(2) Forthe5MVPs under investigation, there exits methylation difference among the commonforensic stains. They may be used as molecular stain markers.(3) The discriminationof blood, semen, saliva, menstrual blood and vaginal fluid can be realized by acombination analysis of3MVPs (MAGE1,14-3-3σ,MCJ) by PDP-MCA. |
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