Temporal Temperature Gradient Gel Electrophoresis (TTGE)
Temporal temperature gradient gel electrophoresis (TTGE) is a single nucleotide polymorphism (SNP) detection method based on DNA melting curve analysis. SNP refers to the existence of two or more variant forms of a base pair in different individuals or populations in the DNA sequence, which is the main source of human genetic diversity. SNP is closely related to many genetic diseases, drug reactions, individual differences, etc., therefore, SNP detection is of great significance for gene diagnosis and therapy.
Principles and Steps of TTGE
The basic principle of TTGE is to use the migration pattern of DNA fragments in a denaturing gel with variable temperature and concentration gradients to distinguish different SNPs. A denaturing gel is a polyacrylamide gel containing denaturants (such as formaldehyde or urea) that can cause DNA double strands to dissociate into single strands at a certain temperature and concentration. TTGE is to create different denaturing conditions by changing the temperature and concentration along the vertical direction in the gel under the action of an electric field. When DNA fragments migrate in the gel, they encounter different denaturing conditions, resulting in their dissociation or reassociation. The dissociated DNA fragments have a slower migration speed than the double-stranded DNA fragments, thus forming different bands on the gel. Different SNPs cause different melting curves (the relationship between the dissociation temperature and concentration and the degree of dissociation) of DNA fragments, thus resulting in bands at different positions. By comparing the band patterns of different samples, the presence or absence of SNPs can be determined.
Table 1. A summary of the steps of TTGE
| Step | Main content | Purpose |
|---|---|---|
| DNA extraction | The DNA samples are extracted from the biological materials (such as blood, tissue, and saliva) using standard methods (such as phenol-chloroform extraction, and column purification). | To obtain the target DNA regions containing the SNPs of interest |
| PCR amplification | The target DNA regions containing the SNPs of interest are amplified by PCR using specific primers. The primers can be designed with or without GC-clamps, which are artificial sequences rich in guanine and cytosine that increase the stability of the DNA duplexes. | To increase the quantity and purity of the DNA samples, facilitating subsequent analysis |
| Gel preparation | The denaturing gel is prepared by mixing acrylamide, bis-acrylamide, urea, and TBE buffer. The gel concentration and urea concentration can be adjusted according to the size and melting temperature of the DNA fragments. The gel is poured into a vertical electrophoresis chamber with a temperature gradient device. | To make a gel that can cause DNA double strands to dissociate into single strands at a certain temperature and concentration |
| Electrophoresis running | The PCR products are mixed with loading buffer and loaded into the wells of the gel. A constant voltage is applied to the gel, and the temperature gradient is set to cover the melting range of the DNA fragments. The electrophoresis is run until the DNA fragments reach their respective melting positions. | To make the DNA fragments migrate in the gel, encounter different denaturing conditions, dissociate or reassociate, forming different bands |
| Staining and visualization | The gel is stained with a fluorescent dye (such as SYBR Green I, ethidium bromide, etc.) and visualized under UV light. The band patterns of different samples are compared and analyzed. | To visualize the DNA fragments on the gel, compare the band patterns of different samples, and determine whether there are SNPs |
Characteristics of TTGE
TTGE is a suitable and efficient method for SNP detection that has the characteristics of high resolution, high sensitivity, high throughput, high reproducibility, and low cost. TTGE can separate DNA fragments with only one base pair difference, thus detecting subtle SNPs. TTGE can detect SNPs in low amounts of DNA samples, such as those obtained from clinical specimens or environmental samples. TTGE can analyze multiple samples simultaneously on a single gel, thus saving time and resources. TTGE can produce consistent and reliable results with minimal variation, as long as the experimental conditions are well controlled. TTGE does not require expensive equipment or reagents, such as fluorescent probes or hybridization ovens, thus reducing the cost of SNP detection.
Applications of TTGE
TTGE has been applied to various fields of research and practice, such as molecular biology, medicine, microbiology, ecology, and forensics. Some examples of the applications of TTGE are:
- Detection of mitochondrial DNA mutations: TTGE can be used to identify mutations in the mitochondrial DNA (mtDNA), which are associated with many diseases and disorders, such as migraine, cyclic vomiting syndrome, Alzheimer's disease, Parkinson's disease, diabetes, and cancer. TTGE can also be used to study the genetic diversity and evolution of mtDNA in different populations and species.
- Identification of arbuscular mycorrhizal fungi: TTGE can be used to distinguish different species and strains of arbuscular mycorrhizal fungi (AMF), which are symbiotic fungi that colonize the roots of plants and enhance their growth and resistance to stress. TTGE can analyze the diversity and composition of AMF communities in different soils and plants.
- Screening of TP53 gene mutations: TTGE can be used to screen for mutations in the TP53 gene, which is a tumor suppressor gene that regulates cell cycle and apoptosis. Mutations in the TP53 gene are involved in many types of cancer, such as breast cancer, colorectal cancer, lung cancer, and ovarian cancer. TTGE can detect TP53 mutations with high sensitivity and specificity.
References
- Cornejo P, et al. Temporal temperature gradient gel electrophoresis (TTGE) as a tool for the characterization of arbuscular mycorrhizal fungi. FEMS Microbiol Lett. 2004 Dec 15;241(2):265-70.
- Vásquez A, et al. Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus. Lett Appl Microbiol. 2001 Apr;32(4):215-9.
- Sørlie T, et al. Mutation screening of the TP53 gene by temporal temperature gel electrophoresis (TTGE). Methods Mol Biol. 2014;1105:315-24.
- Jones BM, et al. Temporal temperature gradient electrophoresis for detection of single nucleotide polymorphisms. Methods Mol Biol. 2009;578:153-65.
- Mohammadi P, et al. Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species. Jundishapur J Microbiol. 2015 Oct 12;8(10):e22249.
- Zhu D, et al. Detection of clonal T-cell receptor-gamma gene rearrangement by PCR/temporal temperature gradient gel electrophoresis. Am J Clin Pathol. 2001 Oct;116(4):527-34.
- Wong LJ, et al. Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis. Electrophoresis. 2004 Aug;25(15):2602-10.
- Nieguitsila A, et al. Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture. J Microbiol Methods. 2007 Jul;70(1):86-95.
