epacadostat synthesis To further explore whether it is the
To further explore whether it epacadostat synthesis is the bisulfite conversion or the HRM amplification that limited the efficiency of amplification of DNA of DNA levels below 1 ng, we performed serial dilutions of bisulfite-modified samples prepared with 1 ng of input genomic DNA. Four different quantities of DNA were amplified based on dilutions of the original 1 ng level of input DNA. Fig. 4 shows that a dilution of 1:2 is sufficient to impair amplification for two of nine samples. Because the bisulfite-modified samples are further reduced in quantity, fewer samples amplify successfully, as can be seen for the 1:16 dilution having only five successful amplifications out of a total of nine samples. Fig. 4 also shows that for the dilutions of 1:2 and 1:4, when amplification is successful the expected TM value is observed for the body fluid tested. The saliva samples at 1:8 dilution show a higher standard deviation (77.50 ± 0.72) when compared with 1:2 dilution (78.50 ± 0.15) and 1:4 dilution (78.25 ± 0.00), possibly due to stochastic effects on amplification and melt analysis when low amounts of DNA are used. The results displayed in Fig.3, Fig.4 demonstrate that samples containing less than 1 ng of input DNA prior to bisulfite conversion may result in impaired amplification due to the presence of low levels of DNA. However, in situations where amplification occurs below 1 ng, distinctions among semen, blood, and saliva may still be possible using HRM analysis. The fact that the melting temperatures do not change when low amounts of DNA are successfully amplified demonstrates that this method is unlikely to give false results that would otherwise lead to a misclassification of a body fluid. To further test the robustness of the method, we explored whether the primers can amplify non-bisulfite-modified DNA. Differences in methylation can be explored accurately only if the DNA is bisulfite modified prior to amplification and HRM. Incomplete bisulfite modification could result in an overestimation of methylation in DNA because an amplicon containing unmodified cytosines, regardless of their methylation status, will have a melting curve similar to a fully methylated amplicon . To determine whether our primers are specific for bisulfite-converted DNA, we performed HRM for the same extracted samples of semen, blood, and saliva before bisulfite conversion (gDNA) and after bisulfite conversion (bDNA). Fig. 2 demonstrates that gDNA samples did not amplify (pink lines) even after 50 cycles. The bDNA amplified properly (blue, orange, and green lines in Fig. 5A) and presented the expected melt curves, with a lower TM for semen when compared with blood and saliva (Fig. 5B). Because some samples contain substances that, when coextracted with DNA, can cause inhibition of the amplification, we determined whether the cleanup steps in the bisulfite conversion reaction are sufficient to remove inhibition. Because humic acid is an inhibitor known to impair amplification by binding to the template DNA , we decided to examine whether the cleanup step can release DNA from that substance. Moreover, to determine whether the PCR efficiency is altered in the presence of humic acid, we also added that inhibitor following bisulfite conversion. PCR efficiency is evaluated by the number of copies of DNA synthesized per cycle (or unit of time). On a graph that displays the amplification curve, such as in Fig. 6A, a steeper slope on the exponential curve means that the PCR was more efficient. Efficiency is 100% when E = 2 (E = 10slope), and it means that the PCR product doubles at each cycle. In the first experiment, humic acid was added before bisulfite conversion to simulate coextraction with DNA. Fig. 6A shows that samples without humic acid (control, thin lines) and samples where the humic acid was added before bisulfite conversion (dashed lines) amplify with similar efficiency. The fact that the steepness of the curves for the control samples and those where humic acid was added before bisulfite treatment are similar allows us to conclude that the cleanup steps performed as part of the bisulfite kit are sufficient to remove humic acid coextracted with DNA.