Several reports have exploited the
Several reports , , , , have exploited the application of HRM for detecting differentially methylated DNA. Even though DNA methylation analysis by HRM cannot determine the methylation status of individual CpGs, it does provide a robust and inexpensive method to differentiate DNA based on the overall methylation of a specific amplicon.
One challenge faced in PCR-based analysis is the potential decrease of amplification efficiency due to inhibition. DNA is often coextracted with substances that can hinder PCR. There are two main mechanisms of PCR inhibition. One mechanism occurs when the inhibitor binds the DNA, and the other mechanism occurs when the inhibitor hinders the catalytic activity of polymerase. When the inhibitor binds to the DNA, it can diminish the processivity of the DNA polymerase or prevent primers from annealing to the template DNA, thereby decreasing PCR efficiency. Moreover, for inhibitors binding to the DNA, a simple cleanup step might not be sufficient to remove the decrease in PCR efficiency. Humic BI2536 is one substance known to inhibit PCR because it binds to the template DNA , .
In this study, we aimed to investigate whether the methylation differences among blood, saliva, and semen for the locus ZC3H12D can be identified using HRM. When pyrosequencing analysis is performed in the locus ZC3H12D, blood and saliva show hypermethylation in comparison with semen . The successful analysis of this locus using HRM would be extremely useful in the identification of the source of DNA samples in forensic investigations. To our knowledge, this is the first time that HRM has been used to discriminate body fluids by exploring differences in DNA methylation.
Materials and methods
Results and discussion High-resolution melt can be used to discriminate among fragments of DNA that present varying degrees of methylation. HRM has the advantage of being cost-effective, besides being a useful alternative/additional method to pyrosequencing protocols, because it permits the analysis of amplicons with sizes larger than 70 bp. From the work of Madi and coworkers , we note that for locus ZC3H12D semen presents low levels of methylation (∼10%), whereas other body fluids have levels of methylation around 100%. The hypomethylation of semen is expected to result in a melt curve with a lower melting temperature when compared with blood and saliva cells. Fig.1, Fig.2 illustrate that DNA from semen presents an average melt temperature of 75.5 °C with a standard deviation of 0.2, which is lower than the values for other body fluids (78.2 and 78.1 °C for blood and saliva, respectively). Our primers amplify a specific genomic region that is hypermethylated in saliva and blood, resulting in amplicons with high GC content. Methylated cytosines are protected from bisulfite conversion and remain as cytosines. Amplification will result in amplicons with a high GC content, which results in higher melting temperature (TM) because higher temperatures need to be reached to break the triple hydrogen bonds that bind guanine to cytosine. In semen, the same region amplified by our primers is hypomethylated. The lack of methyl groups in the cytosines from semen DNA causes them to convert to uracil through the bisulfite reaction. Those uracils are amplified as thymines and result in an amplicon with lower GC content, resulting in melt curves with lower TM values. The differences in TM are visible in Fig.1, Fig.2. To determine the sensitivity of this method, we performed serial dilutions of DNA samples from blood, saliva, and semen to obtain inputs of DNA to bisulfite conversion of 1, 0.5, and 0.25 ng. Those samples were amplified, and the TM value was quantified by HRM. Fig. 3 shows that for inputs of DNA lower than 1 ng, some samples fail to amplify even when the amplification is extended for 50 cycles. It seems that 1 ng of genomic DNA is the minimum amount to input to bisulfite conversion to guarantee amplification using this protocol for HRM analysis.