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In the deep exploration of contemporary molecular biology, complementary DNA (cDNA) has become the core factor to promote the development of multi-dimensional research with its excellent characteristics. With the deepening study of gene expression regulatory network, functional genomics framework and molecular pathological mechanism of diseases, the limitations of traditional research strategies in the face of complex life processes are increasingly prominent.
As the product of mRNA reverse transcription, cDNA accurately and specifically presents the whole picture of gene expression of cells in a specific physiological or pathological state, which provides a key entry point for unlocking the complicated molecular events in the life process. It has been widely and deeply applied in many frontier academic fields such as basic scientific research, translational medicine and biotechnology development, showing irreplaceable important value. In particular, transcriptome sequencing based on cDNA has become a powerful tool to systematically study gene expression patterns and transcript diversity.
This paper introduces the application of cDNA in gene cloning and expression, PCR, disease research and drug development, discusses its future development trend in Qualcomm quantity and single cell analysis, integration and innovation with other technologies.
cDNA in Gene Cloning and Expression Studies
Gene cloning and expression research is the core content of molecular biology, which is very important for understanding gene function, disease mechanism and biotechnology application. CDNA is a copy of DNA produced by mRNA reverse transcription, which represents the gene expressed by cells at a specific time and under certain conditions. Because cDNA does not contain introns, it has unique advantages in gene cloning and expression research.
Full-length gene cloning: cDNA library is an important resource for full-length gene cloning. By screening the cDNA library, the complete coding sequence of a specific gene can be obtained. Compared with cloning genes from genomic DNA, using cDNA can avoid the interference of introns and simplify the cloning process. In addition, cDNA library can also be used to discover new genes and transcripts.
Construction of gene expression vector: Inserting cDNA into an appropriate expression vector is a key step to realize gene expression. Expression vectors usually contain regulatory elements such as promoters, enhancers and terminators, which can drive the transcription and translation of cDNA in host cells. Commonly used expression vectors include plasmids, viral vectors, etc., and the appropriate vectors are selected according to the research purpose and host cells.
Protein expression and function research: The recombinant protein can be produced by introducing cDNA into host cells for expression, which can be used to study the structure and function of protein. Through the purification and analysis of recombinant protein, we can deeply understand its biological activity, interaction and mechanism. In addition, the cDNA expression system can also be used for drug screening and development.
cDNA has important application value in gene cloning and expression research. By preparing high-quality cDNA library and selecting appropriate expression vectors, genes can be cloned and expressed effectively, and the function and regulation mechanism of genes can be deeply studied. With the continuous development of technology, cDNA technology will play a more important role in biomedicine, agriculture and industry.
cDNA expression in mammalian cells (Udo et al., 2015)
Role of cDNA in PCR and qPCR
Polymerase chain reaction (PCR) and its derivative real-time fluorescence quantitative polymerase chain reaction (qPCR) are important tools for studying gene expression and detecting gene mutation. cDNA plays a key role in these two technologies, which greatly promotes the progress of biological research. The application of cDNA in qPCR and PCR will be described in detail below.
Detection of gene expression: PCR can be used to detect the expression of specific genes, by extracting mRNA from different tissues or cells in different physiological states, reverse transcription into cDNA, and PCR amplification with primers for specific genes. If the target band is amplified, it means that the gene is expressed in the corresponding tissue or cell. This method can detect gene expression quickly and sensitively, and provides a powerful means for studying the spatio-temporal expression pattern of genes.
Mutation detection: cDNA can also be used to detect gene mutation. When the gene mutates, the mRNA sequence produced by its transcription will also change accordingly, and the cDNA obtained by reverse transcription will also carry mutation information. By designing suitable primers, amplifying cDNA by PCR, and then sequencing the amplified products or other molecular biological analysis, gene mutation can be detected.
Quantitative analysis of gene expression: qPCR is a technology that can quantitatively analyze nucleic acid. In the study of gene expression, the application of cDNA in qPCR is particularly important. After extracting mRNA from cells or tissues and reverse transcription into cDNA, qPCR reaction was carried out with cDNA as template. In the process of qPCR, the change of fluorescence signal is directly proportional to the amount of amplified products. By detecting the intensity of fluorescence signal, the expression level of the target gene can be accurately quantified. Researchers can compare the expression differences of the same gene in different samples, or analyze the expression changes of the same gene under different treatment conditions.
MicromiRNA detection: MicroRNA is a kind of noncoding RNA with short length, which plays an important role in gene expression regulation. Because of the short length of miRNA, the traditional detection methods have some limitations. The expression level of miRNA can be effectively detected by cDNA combined with qPCR technology. Firstly, the miRNA was reverse transcribed into cDNA, and then the specific primers for miRNA were designed for qPCR amplification and quantitative analysis. This method has high sensitivity and specificity, and can accurately detect the expression changes of miRNA in different physiological and pathological States, which provides an important means for studying the biological function and disease diagnosis of miRNA.
Pathogen detection and quantification: cDNA-qPCR technology is also widely used in infectious disease diagnosis and pathogen research. For RNA viruses, such as influenza virus, Covid-19, etc., firstly, the RNA of the virus is extracted and reverse transcribed into cDNA, and then the viral nucleic acid is quantitatively detected by qPCR technology. This method can quickly and accurately detect the presence of pathogens and quantitatively analyze the load of pathogens, which is of great significance for early diagnosis, disease monitoring and treatment effect evaluation.
qPCR results representing expression levels are shown as a bar graph (Schibler et al., 2009)
cDNA Libraries and Their Importance
cDNA library refers to a clonal population formed by connecting cDNA fragments formed by reverse transcription of all mRNA transcribed by a specific tissue or cell of an organism with appropriate vectors and introducing them into recipient bacteria.
Biotechnology Application
Protein production: The large-scale production of protein can be realized by obtaining the cDNA encoding a specific protein from a cDNA library, cloning it into an expression vector and introducing it into a suitable host cell (such as Escherichia coli, yeast, etc.). This has important applications in the field of biopharmaceuticals, such as the production of pharmaceutical proteins such as insulin and growth hormone.
Gene therapy: cDNA library provides gene source for gene therapy. In gene therapy, it is necessary to introduce normal genes into patients to correct the function of defective genes. By screening suitable genes from cDNA library, it can be used in the research and application of gene therapy.
Research on Evolution and Phylogeny
The cDNA libraries of different species contain their own gene information. By comparing the cDNA libraries of different species, we can study the evolutionary relationship and phylogeny between species. Analyzing the cDNA libraries of different mammals is helpful to understand their gene evolution and differentiation in the process of evolution and provide an important basis for the study of evolutionary biology.
Application of Agriculture and Animal Husbandry
cDNA library also plays an important role in agriculture and animal husbandry. By constructing cDNA libraries of crops and livestock, genes related to excellent traits, such as disease resistance, stress resistance and high yield, can be screened out. By introducing these genes into recipient varieties, new varieties with excellent characters can be cultivated and the production efficiency and quality of agriculture and animal husbandry can be improved.
Difference between the cDNA microarray and RT-PCR in DNA-damage repair and transcription genes (Dai et al., 2013)
cDNA in Disease Research and Drug Development
In the field of modern medicine, cDNA has become a key tool in disease research and drug development. It plays an irreplaceable role in revealing disease mechanism, finding diagnostic markers and developing new drugs.
Study on Disease Mechanism
Detection of gene mutation and variation: cDNA can be used to detect mutation and variation in genes. The reverse transcription process converts mRNA into cDNA, so that the coding region can be directly sequenced and analyzed. In some hereditary diseases, the mutation of specific genes is the root cause of the disease. These pathogenic mutations can be accurately identified by sequencing the cDNA of patient samples.
Signal pathway research: Intracellular signal pathways play a key role in maintaining normal physiological functions of cells and the occurrence and development of diseases. cDNA can be used to study the expression and interaction of genes in signal pathway. By constructing cDNA library of genes related to specific signal pathways and screening their functions, we can find new signal molecules and regulatory mechanisms.
Disease Diagnosis Discovery
Prognostic evaluation markers: In addition to early diagnosis, it is also very important to evaluate the prognosis of the disease for making personalized treatment plans. cDNA technology can be used to screen gene markers related to disease prognosis. In the study of breast cancer, through the cDNA analysis of a large number of patients’ tumor tissues, it is found that the expression level of some specific genes is closely related to the recurrence risk, survival rate and other prognostic indicators of patients.
Disease Typing Marker
There are different subtypes of many diseases, and the treatment methods and prognosis of different subtypes may be different. cDNA analysis can be used for molecular typing of diseases. In the study of lung cancer, lung cancer is divided into different molecular subtypes by analyzing the gene expression profile of cDNA in tumor tissue, and each subtype has unique gene expression characteristics and biological behavior. This molecular typing helps doctors to make more accurate personalized treatment plans for patients.
Genome-scale RNAi library construction in S. cerevisiae (Chen et al., 2020)
Drug Development
Drug screening and evaluation: cDNA library can be used for drug screening. Drugs are applied to cell models or animal models that express specific cDNA, and the effects of drugs on gene expression and cell function are observed, so as to screen out drugs with potential therapeutic effects. At the same time, cDNA technology can also be used to evaluate the efficacy and safety of drugs. In the clinical trial of drugs, the effect of drugs on gene expression was evaluated by analyzing the cDNA expression profiles of patients before and after treatment, and whether the drugs achieved the expected therapeutic effect and whether there were potential toxic and side effects.
Gene therapy drug development: cDNA is an important basis for gene therapy drug development. The principle of gene therapy is to introduce normal genes or therapeutic genes into patients to correct defective genes or regulate abnormal gene expression. By cloning the cDNA of therapeutic gene into a suitable vector, such as a viral vector or a non-viral vector, it can be introduced into the target cell. In the gene therapy of some hereditary immunodeficiency diseases, the cDNA of normal immune-related genes is introduced into the hematopoietic stem cells of patients through vectors, so that patients can restore normal immune function.
With the continuous progress of technology, cDNA technology is expected to play a greater role in disease research and drug development. Combining artificial intelligence and machine learning technology, more accurate analysis and interpretation of cDNA data will help to discover more disease mechanisms and drug targets, and promote the development of personalized medicine.
Future Trends in cDNA Technology
cDNA technology has become the core tool to study gene expression, function and biomedical application. With the continuous progress of science and technology, cDNA technology is developing towards higher accuracy, sensitivity and flux, and it is also expanding its application in emerging fields. The following will discuss several important trends of cDNA technology in the future.
Super Qualcomm Quantity and Single Cell Analysis
Super high-throughput sequencing combined with cDNA technology: With the continuous reduction of sequencing cost and the rapid development of sequencing technology, Super high-throughput sequencing will be more closely combined with cDNA technology. The traditional sequencing method can only analyze a few samples at a time, but the future super high-throughput sequencing platform can sequence a large number of cDNA samples in a short time, which greatly improves the research efficiency. In large-scale gene expression profile research, thousands of cell samples can be sequenced at the same time, and the differences of gene expression in different cell States can be fully understood. This will help to find new disease markers and therapeutic targets, and promote the development of personalized medicine.
In-depth application of single cell cDNA analysis: Single cell cDNA analysis technology will be more widely used in the future. There are significant heterogeneity among individual cells, and traditional population cell analysis methods often cover up these differences. Single cell cDNA technology can reveal the unique gene expression pattern of each cell, providing an unprecedented perspective for in-depth understanding of cell differentiation, development and disease mechanism.
The rapid development of Whole Genome Sequencing (WGS) technology provides a new perspective for cDNA research, which can provide an important reference for the construction of cDNA library. By comparing the cDNA sequence with the whole genome data, researchers can accurately identify the source genes of transcripts, discover new alternative splicing events and non-coding RNA, and further enrich the understanding of the complexity of gene expression. This method of multi-omics integration analysis provides a powerful tool for precision medicine and functional genomics research.
Integration and Innovation with Other Technologies
cDNA combined with CRISPR gene editing technology: CRISPR gene editing technology has become a powerful tool for biological research, and it will be combined with cDNA technology to achieve more accurate gene function research in the future. By combining cDNA with CRISPR system, we can accurately regulate the expression of specific genes in cells, and at the same time analyze the downstream cDNA to deeply understand the function and regulatory network of genes.
Combining cDNA with protein genomics: Combining cDNA technology with protein Genomics can realize comprehensive analysis from gene to protein. Although cDNA technology can provide information about gene expression, protein is the direct executor of cell function. By integrating cDNA and protein omics data, the relationship between gene expression and protein function can be further understood.
Better study the biodegradation mechanism and metabolic process through cDNA (Hu et al., 2024)
Conclusion
In the field of biotechnology, cDNA has a very wide and critical application. In the research and development of genetic engineering drugs, medical proteins can be obtained by obtaining the cDNA of corresponding cells and cloning it into expression vectors, introducing it into host cells for large-scale expression, and finally purifying it. In gene therapy research, cDNA can be used as the source of therapeutic genes, and specific cDNA can be introduced into patients with the help of vectors to make up or correct the function of defective genes.
For more related content, you may refer to the following articles:
- Overview of cDNA Synthesis
- cDNA vs. gDNA: Structure, Function, Biotech Apps, and Why Choose cDNA
- Exploring cDNA: Introduction, Synthesis, gDNA Comparison, and Overcoming Challenges
- Reverse Transcription: An In-Depth Exploration of Structure, Function and Application
- cDNA Library is Worth Your Trust
References
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- Schibler L, Gibbs L., et al. "New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling." PLoS One. 2009 4(10):e7633 https://doi.org/10.1371/journal.pone.0007633
- Kang H, Hata A. "Quantitative Real-Time PCR Analysis of MicroRNAs and Their Precursors Regulated by TGF-β Signaling." Methods Mol Biol. 1344:313-23 https://doi.org/10.1007/978-1-4939-2966-5_20
- Chen Y, Guo E., et al. "Advances in RNAi-Assisted Strain Engineering in Saccharomyces cerevisiae." Front Bioeng Biotechnol. 2020 8:731 https://doi.org/10.3389/fbioe.2020.00731
- Dai ZJ, Gao J., et al. "Targeted inhibition of mammalian target of rapamycin (mTOR) enhances radiosensitivity in pancreatic carcinoma cells." Drug Des Devel Ther. 2013 7:149-59 https://doi.org/10.2147/dddt.s42390
- Hu Z, Qian C., et al. "Comprehensive toxicological, metabolomic, and transcriptomic analysis of the biodegradation and adaptation mechanism by Achromobacter xylosoxidans SL-6 to diuron." Front Microbiol. 2024 15:1403279 https://doi.org/10.3389/fmicb.2024.1403279