Confirmation Sensitive Gel Electrophoresis

Human genetic diseases refer to a class of diseases caused by genetic factors that have the characteristics of heredity, diversity, complexity, and universality. The diagnostic methods of human genetic diseases refer to various technologies and means used to detect and identify human genetic diseases, which are the basis and key to the prevention and treatment of human genetic diseases. Confirmation sensitive gel electrophoresis (CSGE) is a rapid screening method for the detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. It has been widely used for mutation screening in genetic disorders and for the detection of single nucleotide polymorphisms (SNPs). CSGE is based on the principle that DNA heteroduplexes, which are formed by hybridizing two different DNA strands, have different conformations and migration rates in a gel matrix than DNA homoduplexes, which are formed by hybridizing two identical DNA strands. CSGE can detect subtle changes in DNA sequences with high sensitivity and resolution and has advantages over other methods such as low cost, a low false positive rate, and a simple procedure.

Principles and steps of CSGE

The basic principle of CSGE is to use the different conformational changes of DNA duplexes at different temperatures, which affect their migration rates in a gel matrix. The difference and connection between CSGE and conventional gel electrophoresis are that CSGE uses a special denaturing gel, which can maintain the partial denaturation state of DNA duplexes within a certain temperature range, while conventional gel electrophoresis uses a non-denaturing gel, which can only maintain the complete denaturation or complete non-denaturation state of DNA duplexes. The common point between CSGE and conventional gel electrophoresis is that they both separate DNA molecules by size in a gel by applying an electric field and then detect them by staining or other methods. The advantage of CSGE is that it can detect small DNA sequence variations, such as single-base substitutions or small fragment insertions or deletions, while conventional gel electrophoresis cannot. The disadvantage of CSGE is that it requires higher control of experimental conditions, such as temperature, voltage, time, etc., while conventional gel electrophoresis is relatively easy to operate.

Table 1. The steps of CSGE

Step Main Content Purpose Precaution
DNA extraction Extract DNA from human genetic disease patients or normal people's samples. Obtain the source material for the target DNA fragment. Ensure the quality and purity of DNA, and avoid contamination and degradation.
Amplification Use specific primers to amplify the target DNA fragment by PCR. Generate enough DNA templates and increase the detection sensitivity. Choose suitable primers, optimize PCR reaction conditions, and control amplification cycles.
Denaturation Denature the amplified DNA samples at a high temperature, then anneal them at an appropriate temperature. Make different sources of DNA single strands re-pair to form duplexes, which may produce incomplete matching hybrid duplexes or complete matching homologous duplexes. Determine the appropriate denaturation temperature and annealing temperature to maximize the ratio of heteroduplexes and homoduplexes.
Electrophoresis Load the denatured DNA samples onto a special denaturing gel, then apply an electric field to separate DNA molecules by size and conformation in the gel. Make heteroduplexes and homoduplexes form different bands on the gel, reflecting the presence or absence of DNA sequence variation. Choose suitable voltage, time, and temperature to keep the gel in a certain degree of denaturation state, and avoid complete denaturation or complete non-denaturation of DNA duplexes.
Staining and analysis Stain the gel with dye to make DNA bands visible, then observe and analyze them with a microscope or other instruments. Judge whether there is DNA sequence variation according to the number and position of bands: if yes, further determine its type and location Choose a suitable dye, make bands clear and distinguishable, avoid over- or under-staining, and use appropriate instruments and software for analysis.

Applications of CSGE in Human Genetic Disease Diagnosis

The application scope and effect of CSGE in human genetic disease diagnosis are very extensive and significant. It can be used to detect various types of DNA mutations, such as single-gene genetic diseases, complex genetic diseases, mitochondrial genetic diseases, etc. CSGE can not only improve the diagnostic accuracy and prevention effect of human genetic diseases but also provide the basis and guidance for personalized medicine and gene therapy.

First, CSGE can be used to screen for pathogenic gene mutations in single-gene genetic diseases, thus providing molecular diagnosis and genetic counseling services for patients. For example, CSGE can be used to screen LDLR gene mutations in familial hypercholesterolemia (FH), which encodes the low-density lipoprotein receptor. These mutations can lead to increased serum cholesterol levels and an increased risk of coronary heart disease. CSGE can detect multiple exonic regions of LDLR gene mutations in one electrophoresis, thus providing an accurate molecular diagnosis and personalized treatment plan for FH patients.

Second, CSGE can be used to detect single nucleotide polymorphisms (SNPs) related to complex genetic diseases, thus providing risk assessment and drug selection services for patients. For example, CSGE can be used to detect BRCA1/2 gene mutations in breast cancer patients, which encode breast cancer susceptibility proteins. Their mutations can cause DNA repair function defects, increasing the risk of breast cancer and ovarian cancer. CSGE can detect multiple regions of BRCA1/2 gene mutations in one electrophoresis, thus providing effective molecular typing and drug sensitivity testing for breast cancer patients.

Third, CSGE can be used to detect pathogenic mtDNA mutations in mitochondrial genetic diseases, thus providing diagnosis, prognosis, family tracing, and genetic counseling services for patients. For example, CSGE can be used to detect common mtDNA mutations in Leber hereditary optic neuropathy, such as G11778A, T14484C, and G3460A. These mutations can cause functional impairment of retinal pigment epithelial cells and optic nerve cells, leading to acute or subacute vision loss. CSGE can detect the presence or absence of these mtDNA mutations in one electrophoresis, thus providing rapid and accurate molecular diagnosis for Leber hereditary optic neuropathy patients.

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For research use only. Not intended for any clinical use.