Gene Mutation and Sequencing

What is Gene Mutation?

Gene mutations, alterations in the genetic code, can have significant impacts on the growth and development of organisms. Often, these mutations result in the loss of the gene's original function, disrupting the coordinated relationship between genes and related metabolic processes. The consequences may manifest as trait variations, abnormal individual development, reduced competitiveness for survival and reproduction, or, in extreme cases, lead to lethal mutations.

Mutation Type Description Mutation Type Description Recommended Reading
Point Mutation Point mutation involves a change in a single base or base pair of DNA, leading to variations in the genetic code. Synonymous Mutation This occurs when a base substitution takes place in the coding sequence of a gene. However, due to codon redundancy, the mutated codon and the original codon may code for the same amino acid, resulting in no alteration to the translated protein.
Missense Mutation In this scenario, a base substitution within the gene's coding sequence changes the codon, leading to the coding of a different amino acid. This alteration causes the polypeptide chain to lose its original function, resulting in abnormalities in the protein.
Nonsense Mutation A mutation in the gene's coding sequence forms one of the three nonsense codons (UAG, UAA, UGA). This mutation results in the premature termination of protein synthesis, leading to a shortened protein. This truncation may potentially impact or entirely disrupt the protein's function.
Insertion-Deletion Mutation (Indel) Indel involves the insertion or deletion of a certain length of nucleotides at a specific position in the genome. Frameshift (fs) This occurs when one or more bases, not integer multiples of 3, are inserted or deleted on the DNA strand. This disrupts the reading frame in the coding region, altering the sequence of bases and leading to changes in the translated amino acids.
Non-Shift Mutation If one or more bases are inserted or deleted in an integer multiple of 3 on a DNA strand, the sequence of bases in the coding region is altered. This results in a change in the translated amino acid without disrupting the reading frame.
Dynamic Mutation Dynamic mutation, also known as unstable trinucleotide repeat sequence mutation, is caused by the amplification of trinucleotide repeats in the gene coding sequence or flanking sequence. The number of repetitive amplifications can increase across generations, showcasing a cumulative mutation effect, hence the term dynamic mutation. Common in various genetic disorders such as Huntington's disease, fragile X syndrome, spinocerebellar ataxia, and ankylosing muscular dystrophy.

Cutting-edge technologies, such as high-throughput sequencing and long-read sequencing, employed by CD Genomics, facilitate the robust analysis of gene mutations. This advanced sequencing approach allows for comprehensive and efficient examination of genetic material, providing valuable insights into the molecular landscape and potential biomarkers associated with various conditions.

Classifying Mutations

Mutations are classified based on their origins into two main types: spontaneous and induced mutations, each arising from distinct causes. Spontaneous mutations encompass those occurring naturally, driven by factors like natural mutagens or errors during DNA replication and repair. On the other hand, induced mutations result from the intentional use of artificial mutagens. Mutagens come in various forms, including physical mutagens like ionizing radiation and ultraviolet light, chemical mutagens such as nitrite and ethidium bromide, and biological mutagens like retroviruses.

Further categorization of mutations is based on the cells responsible for their production. Somatic cell mutations and germ cell mutations are two primary types. Somatic cell mutations, arising in non-reproductive cells, do not directly transfer to the next generation. In contrast, germ cell mutations, occurring in reproductive cells, possess a higher likelihood of being inherited by offspring.

Another perspective for categorization considers the regions producing mutations. At the cellular level, chromosomal mutations involve alterations in the number and structure of chromosomes within a cell. Meanwhile, at the molecular level, genetic mutations involve changes in the composition and sequence of bases within genes.

Features of Mutation

The perception of mutations as harmful or beneficial is relative, and in certain instances, their effects can transform. For example, a resistance mutation can be considered favorable, as seen in crops with dwarf mutants thriving in windy and high-fertilizer environments.

  • The Prevalence and Rarity of Gene Mutations

Gene mutations are ubiquitous in the biological world, occurring in both lower and higher organisms through natural and artificial means. However, in their natural state, mutations are exceptionally rare, with wild-type genes exhibiting a very low mutation rate.

  • Randomness and Undirected Nature of Gene Mutations

a. Randomness in Site: Gene mutations can occur in somatic cells and germ cells, with the former generally not passed on to offspring and the latter potentially inherited. Mutations may occur in different parts of the same DNA molecule or in distinct DNA molecules within a cell.

b. Randomness in Time: Gene mutations can take place at any stage of an individual's biological development and may be more prone to occur in aging individuals, such as the elderly being susceptible to skin cancer.

c. Non-Directionality: Gene mutations can happen in multiple directions, resulting in various allele forms within a gene.

  • Repeatability and Reversibility of Gene Mutations

a. Repeatability: Mutated genes may undergo additional mutations independently under specific conditions, forming new allele forms at a certain frequency.

b. Reversibility: The direction of gene mutation is reversible.

  • Parallelism of Gene Mutation

Closely related species often undergo similar gene mutations due to their relatively close genetic bases.

Influencing Factors

  • Physical Factors: Exposure to gamma rays, ultraviolet rays, etc.
  • Chemical Factors: Presence of base analogs, aflatoxin, etc.
  • Biological Factors: Impact of certain viruses and bacteria, etc.
  • Alteration of the number, order, and type of deoxyribonucleotides within a gene, leading to changes in genetic information.

Mutation Detection Technology


Amplification Refractory Mutation System PCR (ARMS-PCR), also referred to as Allele-Specific PCR (AS-PCR), employs a sophisticated approach to control allele-specific extension through the strategic design of 3' end primers. By integrating this primer design with the Taqman probe method, the technology accurately identifies both wild-type alleles and mutations.

In the ARMS PCR process, distinctive nucleotides are incorporated at the 3' end of the two upstream primers corresponding to each allele, with one primer specific to the wild-type and the other to the mutant. During amplification, the upstream primer that lacks a perfect match with the template fails to form complementary base pairs, leading to mismatches, blocked extension, and the absence of PCR product generation. In contrast, the primer system that precisely matches the template successfully amplifies the corresponding PCR products. The fluorescent groups attached to the Taqman probe generate detectable signals, allowing for genotype confirmation through the analysis of fluorescence data. This meticulous approach ensures the accurate identification of wild-type alleles and mutant genes in the examined samples.

Mutant Enrichment PCR

Mutant Enrichment PCR is a two-step amplification process designed for the targeted enrichment of mutated EGFR genes. In the initial step, the wild-type EGFR gene undergoes selective digestion using a restriction endonuclease. This process enriches the mutated EGFR gene, paving the way for the second PCR amplification. Subsequently, the PCR products are detected through electrophoresis, and the determination of EGFR gene mutation status is made based on specific characteristics of the PCR products, such as size or presence/absence.

Compared to the direct sequencing method, Mutant Enrichment PCR exhibits heightened sensitivity and specificity in detecting EGFR activation mutations. Remarkably, it can discern a mutated gene even in the presence of 10^3-10^4 wild-type copies. However, it is essential to acknowledge certain drawbacks inherent in this method. The requirement for two PCR amplifications and enzyme digestion renders the procedure intricate, time-consuming, and susceptible to contamination. Despite these challenges, its enhanced sensitivity and specificity make Mutant Enrichment PCR a valuable tool for accurate detection of EGFR gene mutations.

Sanger Sequencing

Sanger sequencing stands out as one of the most widely employed methods for mutation detection. Its fundamental principle revolves around utilizing DNA polymerase, primers, dNTP, and the strategic omission of a random ddNTP during the PCR amplification process. This ddNTP, acting as a substitute for dNTP, participates in the DNA strand synthesis reaction, becoming randomly incorporated into the DNA strand at specific positions, thereby hindering the continuous extension of the DNA strand.

Through this approach, a series of DNA fragments varying in lengths are generated. These fragments are subsequently fluorescently labeled and subjected to electrophoretic separation, resulting in distinct fluorescent bands. The ultimate outcome is the identification of the DNA fragment containing the mutation site. Sanger sequencing's precision lies in its ability to produce a comprehensive profile of DNA fragments, allowing for the accurate localization of mutations within the sequenced DNA strands.

Pyrophosphate Sequencing

Pyrophosphate sequencing represents a cutting-edge advancement in DNA sequence analysis technology. The underlying principle involves a distinctive sequencing approach where, in each sequencing reaction cycle, only a single type of dNTP is introduced. If this specific dNTP successfully pairs with the template, the polymerase incorporates it into the newly synthesized DNA strand, liberating the pyrophosphate group (PPi). Through a cascade of enzymatic chemiluminescence reactions, PPi undergoes conversion into a visible signal, the intensity of which is directly proportional to the number of nucleotides incorporated in the reaction.

This technology allows for the rapid and precise determination of short target fragments without the need for electrophoresis or fluorescent labeling of DNA fragments. Despite its efficiency, it is worth noting that pyrophosphate sequencing has yet to attain the same level of popularity as direct sequencing. This is primarily attributed to its limited read length and susceptibility to contamination. Nevertheless, its unique methodology positions it as a promising alternative for certain applications in DNA sequence analysis.

Next-Generation Sequencing (NGS)

Advancements in Next-Generation Sequencing (NGS) have propelled it as a transformative force, offering three key advantages over traditional Sanger sequencing. These advantages include high throughput, the substitution of bacterial cloned DNA fragments with versatile DNA libraries, and the elimination of electrophoresis from the sequencing process. This paradigm shift significantly reduces the time and labor costs associated with sequencing.

Recommended reading: Illumina Next-Generation Sequencing (NGS): Principles and Workflow.

Over the past two decades, NGS has garnered widespread popularity, particularly for its pivotal role in identifying numerous mutations and chromosomal variants linked to diseases. In the realm of disease-related gene mutations, NGS has ushered cancer research and clinical diagnosis into the genomic era, realizing the vision of personalized medicine based on tumor mutation characteristics.

NGS applications in cancer clinics encompass a spectrum of techniques, including whole-genome sequencing (WGS), whole-exome sequencing (WES), targeted gene panel sequencing (TS), transcriptome sequencing, epigenetic sequencing, and single-phase sequencing. Distinctions among WGS, WES, and TS primarily arise from their coverage levels. WGS comprehensively covers the entire genome at a rate of approximately 95%-98%, whereas WES and TS selectively capture and sequence DNA fragments from whole exomes or specific target genes, respectively. Given that only about 2% of the human genome comprises coding sequences, WES and TS strategically enhance sequencing depth in a cost-effective manner, preserving accuracy and efficiency.

The majority of commercially adopted cancer-related NGS applications leverage the TS method to acquire sequences of cancer-related genes or glean epigenetic information. The continuous evolution and widespread adoption of cancer-related NGS detection theories and technologies have extended its practical applications across various clinical phases. These applications now include early cancer screening, detection of postoperative minimal residual disease (MRD), and concurrent diagnostics for patients grappling with advanced cancers.

Please read our article Next-Generation Sequencing for Cancer Biomarker Discovery.

Single-Molecule Long-Read Sequencing Technology

The innovative single-molecule long-read sequencing technology holds significant advantages in the detection of gene mutations, particularly excelling in analyzing "difficult sequences" that pose challenges for conventional techniques. These may include GC-rich short tandem repeats (STRs) and STRs comprised of repetitive sequence motifs that deviate from the reference genome.

A key strength of single-molecule long-read sequencing lies in its ability to bypass PCR amplification, allowing for comprehensive coverage of the entire genome region, especially in repetitively amplified areas. This approach ensures a thorough exploration of the entire repeat amplification region without the limitations associated with PCR. Moreover, the technology delivers more comprehensive results, encompassing the entirety of repeat sequences, details on amplified repeat lengths, repeat unit numbers, and their distribution.

The high resolution of this technology is particularly noteworthy as it provides precise information about interrupted sequences near disease-causing repeats. These interruptions can serve as valuable markers for disease treatment and prognosis. As a result, single-molecule long-read sequencing technology is anticipated to play a pivotal role in molecular diagnostics related to STR amplification abnormalities. Its application promises enhanced sensitivity and specificity for dynamic diseases, offering patients and their offspring more detailed diagnostic and therapeutic insights.

For Research Use Only. Not for use in diagnostic procedures.
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