# DMSO Improves the Ski-Slope Effect in Direct PCR
## 3 Results and Discussion
### Reducing of Ski-Slope Effect by DMSO in Direct PCR  
We investigated the potential of DMSO in the reduction of ski-slope effect and enhancement of amplification in direct PCR system using Prep-n-Go buffer. We observed that DMSO effectively improved amplification of 2800M DNA and 1.2 mm-punched saliva samples (Figure 2). In addition, even in direct PCR systems, DMSO improved the amplification of large-sized DNA sequences but not small-sized DNA sequences, as shown in Figure 1.  
Figure 1: Effect of dimethyl sulfoxide (DMSO) on multiplex PCR amplification kit. (**A**) Amplification of 2800M DNA (2 ug) with dose-dependent concentrations of DMSO (0, 1, 2.5, 3.75, and 5%, _v/v_) using the GlobalFilter Kit. (**B**) Amplification of 2800M DNA (2 ug) with appropriate concentration of DMSO (3.75%, _v/v_) using the GlobalFiler kit. Amplification yield of 2800M DNA was compared in each of small- and large-sized DNA amplified sequences. Intensity of amplified PCR product was measured by Image J software. Figure 2: Effect of dimethyl sulfoxide (DMSO) in direct PCR performed using the GlobalFiler kit with Prep-n-Go buffer (2 uL/25 uL PCR reaction volume). Amplification yield with DMSO examined in 2800M DNA (**A**) and 1.2 mm-punched saliva samples (**B**) and compared in each of small- and large-sized DNA amplified sequences. Intensity of amplified PCR product was measured by Image J software. To further identify these amplification patterns, we quantified the DNA amplified in the direct PCR system using large (amplicon size, 214 bp) and small (amplicon size, 80 bp) autosomal DNA fragments targeted by the Quantifiler Trio DNA Quantification Kit. These sequences can be used as indicators of DNA degradation by comparing the ratio of their quantification results [23]. We confirmed that the amplification of large autosomal DNA was reduced by Prep-n-Go buffer in direct PCR (Figure 3C), which in turn is associated with the ski-slope effect. However, we found that DMSO alleviated this issue, even in 1.2 mm-punched saliva samples (Figure 3C,D). Moreover, we confirmed that the amplification of small autosomal DNA was increased by Prep-n-Go buffer in direct PCR but slightly decreased by DMSO treatment (Figure 3A,B). Based on the results of PCR amplification enhanced by DMSO in direct PCR, we next examined whether DMSO could reduce the ski-slope effect in direct PCR with 50 Korean buccal samples using the OC card. In the STR genotyping kit, the ski-slope effect is generally shown as a decreasing aspect according to larger DNA amplification sizes of STR locus on the same color channel, such as blue dye (6-FAM (90.5-384.5 bp): D3S1358, vWA, D168539, CSF1 PO, and TPOX), green dye (VIC (108.5-347.5 bp): D8S1179, D21S11, and D18S51), yellow dye (NED (75.0-380.0 bp): D2S441, D19S433, TH01, and FGA), red dye (TAZ (83.5-444.0 bp): D22S1045, D5S818, D13S317, D7S820, and SE33), and purple dye (SID (80.0-355.5 bp): D1 OS1248, D1S1656, D12S391, and D2S1338) [24]. We observed that direct PCR with Prep-n-Go buffer alone led to a decrease in the RFU ratio of STR genotyping according to a large-sized STR locus in each color channel (Figure 4). However,  
Figure 3: Quantitative analysis of small or large autosomal DNA by dimethyl sulfoxide (DMSO) amplified using direct polymerase chain reaction (PCR). Quantitative analysis of the amplified DNA was performed using small or large autosomal DNA (amplicon size: 80 or 214 bp, respectively) included in the Quantifiler Trio DNA Quantification Kit in 2800M DNA (**A,C**) and 1.2 mm-punched saliva samples (**B,D**). Data represent mean \(\pm\) standard deviation. we confirmed that the RFU ratio was improved by addition of DMSO along with Prep-n-Go buffer in direct PCR. In particularly, DMSO more significantly improved the RFU ratio in STR locus of large-sized DNA sequences than in small-sized DNA sequences.