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Denaturing High Performance Liquid Chromatography

2024-01-19 14:53

Basic Principles

Raising the working temperature (column temperature) causes DNA fragments to denature, and partially denatured DNA can be eluted with lower concentrations of acetonitrile. Due to the different untwisting characteristics between heterologous double stranded (mismatched) DNA and homologous double stranded DNA, heterologous double stranded DNA is more variable under the same partial denaturation conditions due to the presence of mismatched regions, and is retained by the chromatographic column for a shorter time than homologous double stranded DNA. Therefore, it is eluted first, resulting in a bimodal or multimodal elution curve in the chromatogram.

technological development

This technology was first established by Oefner in 1995, and currently the most widely used and easy to operate system worldwide is the WAVE nucleotide fragment analysis system developed by Transgenomic in the United States. It separates and analyzes nucleic acid fragments using a unique DNA chromatography column - DNASep column.


As a new type of gene mutation screening technology that has only been established and rapidly developed in recent years, it can be automated, high-throughput, and does not require PCR primer modification, purchase of special reagents, detection of marker signals, or other sample processing except for PCR. However, many existing DNA mutation analysis techniques such as single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) cannot meet this requirement. DHPLC has the advantages of high-throughput detection, high automation, high sensitivity and specificity, wide detection range of DNA fragments and length changes, and relatively low cost. Compared with traditional methods such as SSCP and DGGE, DHPLC has many advantages. The results of SSCP are influenced by factors such as blood sample quality and extraction methods, and require gel electrophoresis and electrophoresis; DGGE requires primers to be labeled as there is radioactive contamination, and both methods are time-consuming and labor-intensive. DHPLC, on the other hand, is highly automated and can automatically take samples, with each sample taking only about 8 minutes to detect. The biggest difference between DHPLC and other methods for detecting DNA mutations is that it can purify DNA fragments. Of course, the limitation of DHPLC is that it can only detect heterozygous mutations, but this can be solved by using a mixed method (i.e. mixing homozygous mutation samples with wild-type samples).

Methodological comparative studies have shown that the sensitivity and specificity of DHPLC can reach 96% to 100%, significantly higher than commonly used mutation detection techniques such as DGGE, CCM, CSGE, SSCP, etc. Currently, only fluorescence single stranded conformation polymorphism analysis (F-SSCP) based on capillary electrophoresis technology can rival DHPLC in sensitivity and specificity. However, key factors such as the design of PCR primers, PCR methods and conditions, collection and sorting temperature, and separation gradient also affect the sensitivity of DHPLC detection.

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