Denaturing High-Performance Liquid Chromatography
Denaturing high-performance liquid chromatography (DHPLC) is a fast, sensitive and efficient chromatographic technique for detecting DNA variations. DHPLC utilizes the denaturation and renaturation characteristics of DNA at different temperatures, and separates and detects DNA by reverse-phase chromatography column. DHPLC can detect DNA variations such as single base substitutions, small fragment deletions or insertions with nearly 100% sensitivity and specificity in a few minutes. DHPLC has the advantages of high throughput, high resolution, low cost, easy operation, etc., and is an ideal method for finding DNA polymorphisms. DHPLC plays an important role and has advantages in the diagnosis and gene therapy of human genetic diseases. It can be used to discover and identify gene variations associated with hereditary cancers, hereditary heart diseases, hereditary nervous system diseases, etc., and provide basis and guidance for clinical diagnosis and treatment.
Principle of DHPLC
The basic principle of DHPLC is to use the denaturation and renaturation characteristics of DNA at different temperatures, and separate and detect DNA by reverse-phase chromatography column. DHPLC uses a special buffer solution, which contains triethylammonium acetate (TEAA), which is an ion-pair reagent for ion-pair reversed-phase liquid chromatography. TEAA can form ion pairs with the surface of DNA molecules, making DNA molecules positively charged, and adsorbed on the non-polar stationary phase with the non-polar alkyl end. When the temperature rises, DNA molecules will undergo partial denaturation, that is, double-stranded DNA will unravel into single-stranded DNA. If there are mismatches or variations in DNA molecules, they will be more easily denatured than normal homologous double-stranded DNA, and their retention time in the chromatography column will be shorter. Therefore, by changing the temperature and the gradient of the buffer solution, the separation and detection of DNA variations can be achieved. DHPLC can use ultraviolet detection or fluorescence detection to monitor the separated DNA samples, and can automatically collect the required DNA fragments.
Steps and Precautions of DHPLC
Table 1. Experimental steps of DHPLC
| Step | Content | Purpose | Precautions |
|---|---|---|---|
| Sample preparation | Extract DNA from blood or tissue, or use synthetic oligonucleotides as samples. | To provide high-quality DNA templates for DHPLC. | Avoid interference from impurities such as proteins, polymers, salts, etc. in the samples. |
| PCR amplification | Design specific primers according to the sequence of the target DNA fragment, and perform PCR amplification. | To provide sufficient amounts of DNA fragments for DHPLC. | Choose appropriate primers and PCR conditions, and avoid interference from non-specific amplification or primer dimers. |
| Slow denaturation | Denature PCR products at 95°C for 5 minutes, then slowly renature them to 25°C at a rate of 0.03°C/second, forming heteroduplexes with heterologous double strands. | To increase the sensitivity and specificity of DHPLC detection. | Control the temperature and rate of denaturation and renaturation, and avoid problems such as DNA fragment degradation or recombination. |
| DHPLC analysis | Inject the slowly denatured PCR products into the DHPLC system with a microsyringe, and use software to predict the optimal analysis temperature and buffer gradient according to the sequence and length of the target DNA fragment, and perform chromatographic separation and detection. | To use the denaturation and renaturation characteristics of DNA at different temperatures, and separate and detect DNA by reverse-phase chromatography column. | Choose the appropriate analysis temperature and buffer gradient, so that different types of DNA double strands can be effectively separated and produce different shapes and positions of elution peaks. |
| Result interpretation | Observe and record the shape and position of the elution peaks in the chromatogram, and screen for mutant samples according to abnormal peak shapes. | To judge whether there are DNA variations in the sample based on the differences in elution peaks in the chromatogram, and determine their approximate location and type. | For unclear or complex mutant samples, DNA sequencing or other methods should be performed to verify and identify the mutation type, avoiding misjudgment or omission. |
Application of DHPLC in diagnosis and gene therapy of human genetic diseases
DHPLC plays an important role and has advantages in the diagnosis and gene therapy of human genetic diseases. It can be used to discover and identify gene variations associated with hereditary cancers, hereditary heart diseases, hereditary nervous system diseases, etc. For example, DHPLC can be used to detect mutations in BRCA1 and BRCA2 genes associated with breast cancer and ovarian cancer, as well as mutations in LDLR, APOB and PCSK9 genes associated with familial hypercholesterolemia. DHPLC can also be used to detect gene variations associated with hereditary metabolic diseases, immune deficiencies, blood diseases, etc. In addition, DHPLC can also be used to evaluate the effect of gene therapy, such as monitoring the expression and integration of exogenous genes in transfected cells. The application of DHPLC not only provides basis and guidance for clinical diagnosis and treatment, but also provides a powerful tool for research in the field of human genetics.
References
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