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KAPA HiFi Uracil+ DNA Polymerase is a modified version of KAPA HiFi DNA Polymerase that is engineered to tolerate uracil residues in bisulfite-treated DNA. Bisulfite treatment of DNA results in the conversion of unmethylated cytosine residues into uracil, while the methylated residues are left unmodified. Enzymes used for library amplification in bisulfite-sequencing workflows must be able to read through uracil residues and tolerate low concentrations of AT-rich DNA. Traditional high-fidelity DNA polymerases are typically not suitable for this type of application as the enzymes stall when a uracil is encountered. KAPA HiFi Uracil+ DNA Polymerase has been developed to read through uracil residues while still retaining the performance benefits of HiFi DNA Polymerase, such as high yields, low-bias, and uniform sequencing coverage.
Benefits of KAPA HiFi Uracil+ Kits
Higher yields and reduced size bias for templates containing uracil compared to other methods
More uniform bisulfite-sequencing coverage and improved representation of AT-rich sequences compared to competitor kits
Figure 1. KAPA HiFi Uracil+ is not inhibited by dUTP. SYBR Green real-time PCR was used to monitor amplification of a 452 bp amplicon over ten-fold dilutions (80 pM – 0.8 fM) with or without 0.2 mM dUTP. KAPA HiFi DNA Polymerase shows typical inhibition by uracil, while KAPA HiFi Uracil+ DNA Polymerase shows no amplification inhibition.
Figure 2. KAPA HiFi Uracil+ is not inhibited by dUTP. SYBR Green real-time PCR was used to monitor amplification of a 452 bp amplicon over ten-fold dilutions (80 pM – 0.8 fM) with or without 0.2 mM dUTP. KAPA HiFi DNA Polymerase shows typical inhibition by uracil, while KAPA HiFi Uracil+ DNA Polymerase shows no amplification inhibition.
Figure 3. KAPA HiFi Uracil+ provides higher yields and minimal size bias. Human whole genome bisulfite-treated libraries were amplified using standard protocols with 12, 14, or 16 cycles and the amplified libraries were analyzed using a Bioanalyzer 2100 High Sensitivity DNA chip. When compared with Agilent Pfu Turbo Cx, KAPA HiFi Uracil+ DNA Polymerase produced much higher yields (left) with very little size bias (right)
Figure 4. KAPA HiFi Uracil+ provides higher yields and minimal size bias. Human whole genome bisulfite-treated libraries were amplified using standard protocols with 12, 14, or 16 cycles and the amplified libraries were analyzed using a Bioanalyzer 2100 High Sensitivity DNA chip. When compared with Agilent Pfu Turbo Cx, KAPA HiFi Uracil+ DNA Polymerase produced much higher yields (left) with very little size bias (right)
See data on even coverage with whole genome bisulfite (WGBS) libraries using KAPA HiFi Uracil+.
Figure 5. KAPA HiFi Uracil+ DNA Polymerase provides improved representation of AT-rich sequences compared to Agilent Pfu Turbo Cx kit. 100 ng of bisulfite-treated library DNA was amplified (8 cycles) with KAPA HiFi Uracil+ DNA Polymerase or Agilent Pfu Turbo Cx.
Improved representation of AT-rich sequences
Greater tolerance to AT-rich regions
Improved bisulfite-sequencing read quality
See data showing better representation of AT-rich sequences using KAPA HiFi Uracil+ compared to Agilent Pfu Turbo Cx.
Figure 6. More even coverage with WGBS libraries. Replicate WGBS libraries were prepared from parasite gDNA, bisulfite-treated, and amplified using KAPA HiFi Uracil+ DNA Polymerase or Agilent Pfu Turbo Cx.
Efficient target enrichment of bisulfite-treated DNA
View data showing efficient amplification of bisulfite-treated DNA by KAPA HiFi Uracil after target enrichment.
Figure 7. Identification of differentially methylated regions using the SeqCap Epi Target Enrichment System. Libraries were prepared from Burkitt’s lymphoma cell line (NA04671) or normal control cell line (CEPH; NA12762) DNA with the KAPA HTP Library Preparation Kit, and subjected to bisulfite-treatment after pre-capture amplification. Capture was performed with a 3.2 Mb SeqCap Epi Choice design, and amplified with KAPA HiFi Uracil+. Differential methylation was observed by the large increase in percent methylation in the lymphoma vs. normal cell line DNA.
