Electrophoresis Based Methods for the Diagnosis of Genetic Disorders

Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and charge. Electrophoresis can be divided into different types, such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., with different resolutions and applications. Electrophoresis plays an important role in analyzing human genetic variation and genetic disease diagnosis because it can detect genetic variations in DNA sequence, such as single nucleotide variations, insertions and deletions, copy number variations, etc. These genetic variations are widespread in the human genome, reflecting the genetic diversity and evolutionary history of humans. At the same time, these genetic variations are also associated with the occurrence and development of many human diseases and, therefore, can serve as markers or targets for diseases.

Confirmation Sensitive Gel Electrophoresis (CSGE)

Confirmation sensitive gel electrophoresis (CSGE) is a method that uses the property of DNA double strands to denature and renature at different temperatures to detect DNA sequence variations. CSGE can analyze DNA heteroduplexes and single strands at low and high temperatures, respectively, to improve the sensitivity and specificity of variation detection. The steps of CSGE include: extracting DNA samples, amplifying target regions, mixing amplified products and control products, performing electrophoresis separation, staining and detection. CSGE has been applied to detect human genetic variations and genetic diseases such as multiple endocrine neoplasia type 1, insulin-dependent diabetes mellitus, breast cancer, etc.

Denaturation Gradient Gel Electrophoresis (DGGE)

Denaturation gradient gel electrophoresis (DGGE) is a method that uses the property of DNA double strands to denature at different concentrations of denaturants to detect DNA sequence variations. DGGE can change the concentration of denaturants in the gel within a certain range, forming a denaturation gradient that causes different DNA fragments to denature at different positions, thus separating DNA fragments containing variations. The steps of DGGE include extracting DNA samples, amplifying target regions and adding GC oligonucleotide tails, performing electrophoresis separation, staining, and detection. DGGE has been applied to detect human genetic variations and genetic diseases such as cystic fibrosis, hereditary nonpolyposis colorectal cancer, Alzheimer's disease, etc.

Temporal Temperature Gradient Gel Electrophoresis (TTGE)

Temporal temperature gradient gel electrophoresis (TTGE) is a method that uses the property of DNA double strands to denature at different temperatures to detect DNA sequence variations. TTGE can change the temperature during electrophoresis within a certain range, forming a temperature gradient that causes different DNA fragments to denature at different positions, thus separating DNA fragments containing variations. The steps of TTGE include extracting DNA samples, amplifying target regions and adding GC oligonucleotide tails, performing electrophoresis separation, staining, and detection. TTGE has been applied to detect human genetic variations and genetic diseases such as breast cancer, lung cancer, Parkinson's disease, etc.

Single-Strand Conformational Polymorphism (SSCP)

Single-strand conformational polymorphism (SSCP) is a method that uses the property of DNA single strands to show different spatial conformations under different conditions to detect DNA sequence variations. SSCP can dissociate DNA double strands into single strands by heat or chemical treatment and then perform electrophoresis under non-denaturing conditions so that different conformations of DNA single strands show different migration rates in the gel, thus separating DNA fragments containing variations. The steps of SSCP include extracting DNA samples, amplifying target regions, denaturing DNA samples, performing electrophoresis separation, staining, and detection. SSCP has been applied to detect human genetic variations and genetic diseases such as cystic fibrosis, hereditary nonpolyposis colorectal cancer, Alzheimer's disease, etc.

Fig.1 The single-strand conformation polymorphism process. (Eva Bagyinszky, 2014)

Conclusion

These four methods are all based on the denaturation property of DNA double or single strands to detect DNA sequence variations. Therefore, they all have a certain sensitivity and specificity and can detect genetic variations such as single nucleotide variations, insertions, and deletions. However, these methods also have some common drawbacks, such as the need to amplify DNA samples and optimize electrophoresis conditions, the inability to directly determine the type and location of variations, the need to combine with DNA sequencing or other methods for verification and identification, etc. With the development and popularization of next-generation sequencing technologies, these methods may be gradually replaced or supplemented. To improve the efficiency and accuracy of these methods or expand their application scope, they can be combined with other methods such as chip technology, fluorescence labeling technology, mass spectrometry technology, etc. In addition, these methods can also be applied to different biological samples, such as blood, saliva, urine, feces, etc., to improve their convenience and feasibility or explore their potential in the microbiome and other fields. In summary, electrophoresis methods still have some value and application prospects in detecting human genetic variation and genetic disease diagnosis, but they also need to be constantly improved and innovated to adapt to the development of science and technology and the needs of human health.

References

1. Suh Y, et al. SNP discovery in associating genetic variation with human disease phenotypes. Mutat Res. 2005;573(1-2):41-53.
2. Claussnitzer M, et al. A brief history of human disease genetics. Nature. 2020;577(7789):179-189.
3. Katsanis N, et al. Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with Bardet-Biedl syndrome. Nat Genet. 2000;26(1):67-70.
4. Balogh K, et al. Genetic screening methods for the detection of mutations responsible for multiple endocrine neoplasia type 1. Mol Genet Metab. 2004;83(1-2):74-81.
5. Quigley L, et al. Molecular approaches to analysing the microbial composition of raw milk and raw milk cheese. Int J Food Microbiol. 2011;150(2-3):81-94.
6. Sheffield VC, et al. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A. 1989;86(1):232-236.
7. Myers RM, et al. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 1985;13(9):3131-3145.