Product introduction

In methylation studies, sulfite treatment converts unmethylated C to U, resulting in the presence of U in the gene fragment. Due to the function of high-fidelity enzyme to repair mismatch, U will be regarded as the wrong base to be cut off during the amplification process, resulting in the failure of the reaction. Kapa Hifi Uracil+ is modified on the basis of Kapa Hifi, so that it will not terminate the reaction when it encounters U in the process of PCR reaction, so as to ensure the normal reaction.

1. Main applications

(1) Amplification of methylated library in second-generation sequencing; (2) Other methylation studies.

2. Technical advantages

(1) The gene fragment containing U can be amplified; (2) Fast and efficient; (3) Less Bias in the product;

(4) High AT/GC template can be amplified; (5) High library coverage.

3. Product features(1) The presence of DUTP had no effect on amplification

　Above: Realtime fluorescent PCR amplification of a system containing Uracil showed no inhibition of Kapa Uracil+ by DUTP. The amplification of 452bp gene was monitored by SYBR Green fluorescent quantitative PCR, with a 10fold gradient dilution (80pm0.8fm) of the template. One group contained 0.2mM DUTP and the other group did not contain DUTP. The reagents used for amplification were Kapa Hifi and Kapa Hifi Uracil+. The results showed that Kapa Hifi was significantly inhibited in the presence of DUTP, while Kapa Hifi Uracil+ was not affected.

(2) Efficient amplification of whole human genome libraries treated with sulfites

　Sulfite treatment is easy to cause DNA damage and reduce the amount of DNA products. Therefore, due to the presence of inhibitors, high A/T content, DNA damage and other factors, negative conditions such as low PCR product quantity, inappropriate fragment size, GC Bias and so on occurred during amplification. To compare Kapa Hifi Uracil+ with Agilent's PFU Turbo CX, we compared the amplified human genome libraries after sulfite treatment to verify the amplification yield and amplification Bias of the library.

A human genome library with fragment length of 200400bp was prepared by Kapa library preparation kit, and the template was 5μg human genome DNA. After sulfite treatment, about 2.7% of untransformed genomic DNA was found for quantification using the Kapa library quantification kit. Genomic DNA treated with undiluted sulfites was amplified with Kapa Hifi UraCil + and Agilent PFU Turbo CX, respectively, and the amplified products were analyzed on Bioanalyzer2100, as shown in the figure below.

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