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Complementary DNA

Complementary DNA (cDNA) is double-stranded DNA synthesized from a single-stranded RNA template, typically messenger RNA (mRNA), through reverse transcription catalyzed by the enzyme reverse transcriptase, an RNA-dependent DNA polymerase.^[1]^[2] The process begins with RNA isolation, followed by priming with an oligo(dT) sequence complementary to the mRNA poly-A tail or random hexamers, enabling the enzyme to extend a DNA strand that is complementary to the RNA template; a second DNA strand is then synthesized to form stable double-stranded cDNA.^[3]^[4] Unlike genomic DNA, cDNA lacks introns and regulatory non-coding sequences, representing only the exons of expressed genes and thus providing a focused template for studying protein-coding regions without eukaryotic splicing complexities.^[5] This intron-free nature has made cDNA essential for recombinant DNA technologies, including gene cloning into prokaryotic vectors for heterologous protein expression, construction of expression libraries, and amplification via polymerase chain reaction (PCR) in reverse transcription PCR (RT-PCR) assays to quantify transcript levels.^[6]^[7] Further applications encompass cDNA microarrays for high-throughput gene expression profiling across thousands of sequences, aiding in toxicogenomics, carcinogen identification, and drug safety evaluations by revealing patterns of transcriptional changes in response to stimuli.^[8]^[9] Since its development in the 1970s, leveraging reverse transcriptase discovered in retroviruses, cDNA synthesis has underpinned advances in functional genomics, enabling the decoding of expressed sequences for disease research, therapeutic protein production, and comparative transcriptomics across species.^[10]^[11]

Definition and Characteristics

Molecular Structure and Formation

Complementary DNA (cDNA) consists of a double-stranded DNA molecule whose sequence is derived from a mature messenger RNA (mRNA) template, lacking introns and thus representing only the exons of the transcribed gene.^[6] Unlike genomic DNA, cDNA features a standard B-form double helix structure with a deoxyribose-phosphate backbone, adenine-thymine and guanine-cytosine base pairing via hydrogen bonds, and thymine substituting for uracil present in the original RNA.^[12] The first strand of cDNA is synthesized as a single-stranded DNA polymer complementary (antisense) to the mRNA, with a 5' to 3' polarity opposite to the RNA template.^[2] The formation of cDNA occurs via reverse transcription, a process catalyzed by reverse transcriptase (RT), an RNA-dependent DNA polymerase originally discovered in retroviruses.^[7] RT initiates synthesis at the 3' end of the mRNA, often primed by an oligo(dT) primer annealing to the poly(A) tail, incorporating dNTPs to extend a complementary DNA strand in the 5' to 3' direction.^[13] This produces a RNA-DNA hybrid, where the nascent cDNA strand displaces the RNA template through RT's helicase-like activity.^[4] Second-strand synthesis follows, typically after partial RNase H-mediated degradation of the RNA strand by the same or a separate enzyme, exposing the first-strand cDNA as a template.^[14] DNA polymerase then synthesizes the complementary second strand, yielding blunt-ended or cohesive-ended double-stranded cDNA ready for cloning or amplification. This ds cDNA mirrors the sense sequence of the original mRNA on one strand, enabling it to serve as a template for protein-coding gene expression in heterologous systems.^[16]

Key Properties and Differences from Genomic DNA

cDNA consists exclusively of sequences derived from mature messenger RNA (mRNA), which has undergone splicing to remove introns, resulting in a DNA molecule that lacks the non-coding intron sequences present in eukaryotic genomic DNA.^[6]^[17] This structure enables cDNA to represent only the protein-coding exons of expressed genes, excluding regulatory elements such as promoters, enhancers, and intergenic regions that comprise a significant portion—often over 98%—of genomic DNA in humans.^[18]^[6] A primary functional property of cDNA is its compatibility for heterologous expression, particularly in prokaryotic hosts like Escherichia coli, where the absence of introns bypasses the need for eukaryotic splicing machinery, allowing straightforward production of eukaryotic proteins from the contiguous coding sequence.^[19] In contrast, genomic DNA clones from eukaryotes would require accurate intron removal, which bacteria cannot perform, often leading to non-functional transcripts.^[5] cDNA libraries reflect tissue- or condition-specific gene expression profiles, capturing only transcribed and processed genes at a given time, whereas genomic DNA encompasses the complete, unchanging genome, including silent or pseudogenic regions.^[19] This selectivity makes cDNA smaller in scale—typically yielding libraries with 10^5 to 10^6 clones for a eukaryotic transcriptome versus billions for full genomic coverage—and more targeted for functional studies of coding potential.^[5] Additionally, cDNA synthesis introduces potential biases, such as underrepresentation of low-abundance transcripts or regions with strong secondary structures in mRNA, which do not affect genomic DNA representation.^[19]

Historical Development

Discovery of Reverse Transcriptase

The discovery of reverse transcriptase, an enzyme capable of synthesizing DNA from an RNA template, occurred independently in 1970 through studies on retroviruses, fundamentally altering understandings of genetic information flow. Howard Temin, working at the University of Wisconsin-Madison with Satoshi Mizutani, identified the enzyme in virions of the Rous sarcoma virus, providing direct enzymatic evidence for Temin's earlier provirus hypothesis, which posited that retroviral RNA genomes are reverse-transcribed into DNA intermediates for integration into host genomes.^[20] ^[21] Concurrently, David Baltimore at the Massachusetts Institute of Technology detected the same activity in Rauscher murine leukemia virus extracts, demonstrating DNA polymerase activity dependent on RNA templates and viral RNA-directed synthesis.^[22] ^[23] Their back-to-back publications in Nature on June 27, 1970, confirmed the enzyme's presence via assays showing incorporation of radiolabeled deoxyribonucleotides into acid-insoluble material using purified viral RNA as template, with optimal activity at 37°C and magnesium ions.^[24] This overturned the prevailing unidirectional interpretation of the central dogma, as articulated by Francis Crick, by establishing a RNA-to-DNA transcription mechanism in nature.^[25] The enzyme's characterization revealed it as a DNA-dependent DNA polymerase with RNA-dependent activity, distinguishing it from conventional polymerases by its lower fidelity and ability to initiate synthesis without a primer in some assays, though later clarified to use tRNA primers in retroviruses.^[26] Temin's work built on indirect evidence from inhibition studies in the 1960s, where actinomycin D blocked retroviral replication without affecting RNA synthesis, implying a DNA intermediate, while Baltimore's approach focused on biochemical fractionation of viral particles to isolate the polymerase.^[27] Skepticism persisted initially due to technical artifacts concerns, but replication in multiple retroviral systems and purification to near-homogeneity validated the findings.^[28] For complementary DNA (cDNA) development, this discovery supplied the essential enzymatic tool; within months, researchers adapted purified retroviral reverse transcriptase for in vitro synthesis of DNA copies from eukaryotic mRNA, marking the inception of cDNA libraries by 1972.^[29] ^[30] Recognition came with the 1975 Nobel Prize in Physiology or Medicine, shared by Temin, Baltimore, and Renato Dulbecco for insights into tumor virus-host interactions, underscoring the enzyme's role in oncogenesis via proviral integration.^[27] Subsequent structural studies confirmed reverse transcriptase's multidomain architecture, including polymerase and RNase H activities for primer removal, enabling efficient cDNA production.^[31] These advances, grounded in empirical viral assays rather than speculative models, laid the causal foundation for synthetic biology applications beyond natural retroviral replication.

Early cDNA Synthesis and Cloning Milestones

In 1972, Inder M. Verma and colleagues at the Massachusetts Institute of Technology synthesized the first complementary DNA (cDNA) copies from eukaryotic messenger RNA (mRNA), using purified rabbit globin mRNA as template and reverse transcriptase from avian myeloblastosis virus (AMV). This marked a pivotal advance following the 1970 discovery of reverse transcriptase, enabling the conversion of RNA sequences into stable DNA for study, though initial products were single-stranded and partial in length due to limitations in enzyme processivity and RNA secondary structure. Progress toward double-stranded cDNA (ds cDNA) occurred in the mid-1970s, with Tom Maniatis, Argiris Efstratiadis, and Fotis C. Kafatos developing methods for second-strand synthesis using Escherichia coli DNA polymerase I on rabbit beta-globin cDNA templates, achieving near full-length copies averaging 600-700 nucleotides by 1976. Concurrently, François Rougeon, Philippe Kourilsky, and Bernard Mach reported the first cloning of a eukaryotic cDNA insert—a rabbit beta-globin sequence—into an E. coli plasmid vector in 1975, demonstrating stable propagation in bacteria and laying groundwork for cDNA libraries. These efforts culminated in 1976 with Maniatis and colleagues amplifying and characterizing cloned ds cDNA of the rabbit beta-globin gene, confirming its fidelity to mRNA via sequencing and hybridization, which facilitated detailed structural analysis of expressed genes without genomic introns. By enabling the isolation of coding sequences from complex eukaryotic transcriptomes, these milestones shifted molecular biology toward recombinant DNA applications, though early yields remained low (often <1% full-length clones) due to inefficiencies in tailing, linker addition, and transformation.

Synthesis Methods

RNA Extraction and Preparation

RNA extraction serves as the foundational step in complementary DNA (cDNA) synthesis, involving the isolation of intact RNA molecules, primarily messenger RNA (mRNA), from biological samples such as cells, tissues, or organisms to ensure faithful reverse transcription. High-quality RNA is essential, as degradation or contamination can lead to incomplete or biased cDNA libraries; for instance, RNA integrity numbers (RIN) above 7 are recommended to minimize fragmentation effects on downstream synthesis efficiency.^[32] Common extraction methods include organic solvent-based approaches like phenol-chloroform extraction using reagents such as TRIzol, which denature proteins and separate RNA into the aqueous phase through phase partitioning, yielding high quantities suitable for low-input samples but requiring careful handling to avoid chemical hazards.^[33] ^[34] Silica-based column purification kits, such as those employing spin columns or magnetic beads, have become prevalent for their speed and scalability, binding RNA under chaotropic salt conditions (e.g., guanidinium thiocyanate) while removing contaminants like proteins, DNA, and phenols; these methods typically recover 70-90% of input RNA with reduced RNase exposure time compared to traditional organic methods.^[34] ^[35] Post-extraction, on-column or solution-based DNase I treatment is critical to eliminate genomic DNA carryover, which could otherwise generate artifactual cDNA strands via non-specific priming during reverse transcription; protocols often specify 10-30 minutes incubation at 37°C followed by inactivation.^[13] Quality control involves spectrophotometric assessment (A260/A280 ratio of 1.8-2.1 indicating purity free of proteins or phenols) and integrity evaluation via agarose gel electrophoresis or automated systems like the Agilent Bioanalyzer, where clear 28S and 18S ribosomal RNA bands confirm minimal degradation.^[36] ^[13] For cDNA applications focused on expressed genes, total RNA is often enriched for polyadenylated mRNA using oligo(dT)-cellulose columns or magnetic beads, which hybridize to the 3' poly-A tails of eukaryotic mRNAs, achieving 1-5% recovery of total RNA as mRNA while depleting abundant rRNA and tRNA; this step enhances representation of coding sequences in cDNA libraries, though it excludes non-polyadenylated RNAs like histones or prokaryotic transcripts.^[37] Modern variants integrate mRNA capture directly into reverse transcription via oligo(dT) primers, bypassing separate purification for total RNA workflows in high-throughput sequencing.^[2] RNase-free practices throughout—such as using diethyl pyrocarbonate (DEPC)-treated water, gloves, and aerosol-resistant tips—are non-negotiable to prevent ubiquitous RNase degradation, with inhibitors like RNasin added during lysis to stabilize RNA yields up to 100-200 μg per gram of tissue.^[13]

Reverse Transcription Process

Reverse transcription is the enzymatic process by which single-stranded complementary DNA (cDNA) is synthesized from a messenger RNA (mRNA) template, utilizing a reverse transcriptase enzyme that functions as an RNA-dependent DNA polymerase.^[3] This step inverts the central dogma of molecular biology by copying RNA into DNA, enabling downstream applications such as cloning and expression analysis.^[4] The enzyme, typically derived from retroviral sources like avian myeloblastosis virus (AMV RT) or Moloney murine leukemia virus (MMLV RT), catalyzes the addition of deoxyribonucleotide triphosphates (dNTPs) complementary to the RNA bases, starting from a primer annealed to the template's 3' end.^[38] AMV RT exhibits thermostability, allowing reactions at 42–70°C to minimize RNA secondary structures and improve specificity, while MMLV RT variants often lack RNase H activity to preserve the RNA-DNA hybrid for subsequent steps.^[39]^[38] The process requires key components including purified RNA template (often poly-A selected mRNA), primers such as oligo(dT) for priming at the poly-A tail, random hexamers for broader coverage, or gene-specific primers for targeted synthesis, along with dNTPs, divalent cations like Mg²⁺, and reaction buffer to maintain optimal pH and ionic conditions.^[40]^[2] Reverse transcriptases possess three core activities: RNA-dependent DNA polymerase for first-strand synthesis, DNA-dependent DNA polymerase for second-strand extension in some contexts, and RNase H for degrading the RNA strand in RNA-DNA hybrids, though truncated versions omit RNase H to yield longer cDNA products.^[38] Reaction efficiency depends on enzyme processivity (ability to synthesize long strands without dissociating, typically 1–10 kb for engineered RTs) and fidelity, with thermostable variants from group II introns achieving higher accuracy than standard retroviral RTs.^[42] In a standard protocol, the reaction initiates with primer annealing at 65–70°C to disrupt secondary structures, followed by cooling to the extension temperature (e.g., 42°C for MMLV or 50°C for AMV), where the RT enzyme extends the primer by incorporating dNTPs at a rate of approximately 10–50 nucleotides per second, producing a first-strand cDNA-RNA hybrid.^[40]^[43] Incubation lasts 30–60 minutes, after which the enzyme is inactivated by heat (e.g., 70–95°C for 5–10 minutes) or EDTA chelation to prevent non-specific activity.^[43] Variability in yield arises from factors like RNA quality, secondary structure, and GC content, with high-temperature reactions reducing biases from stable hairpins but potentially introducing thermal degradation.^[44] Recent engineered RTs, such as fusion proteins with group II intron elements, enhance processivity up to full-length transcripts and fidelity by incorporating proofreading mechanisms, addressing limitations in traditional viral-derived enzymes.^[45]^[46]

Second-Strand Synthesis and Amplification Techniques

Second-strand synthesis in complementary DNA (cDNA) production converts the single-stranded RNA-DNA hybrid formed during reverse transcription into double-stranded DNA (dsDNA), enabling downstream applications such as cloning, sequencing, and library construction. This step typically follows first-strand synthesis, where mRNA serves as the template for reverse transcriptase to generate complementary cDNA. Traditional protocols employ RNase H to create nicks in the RNA strand of the hybrid, generating short RNA primers that E. coli DNA polymerase I extends to synthesize the second strand via nick translation, often supplemented by E. coli DNA ligase to seal nicks and improve yield for longer transcripts.^[47]^[48] An alternative classical approach involves forming a hairpin loop at the 3' end of the first-strand cDNA, which self-primes second-strand synthesis using DNA polymerase, though this method risks incomplete extension and bias toward shorter fragments. More efficient enzymatic strategies utilize high-processivity polymerases like T7 DNA polymerase, which leverages its strong 3' exonuclease activity to displace RNA primers and synthesize full-length second strands, particularly when primed with oligo(dA) annealed to poly(dT) tails, yielding longer clones from polyadenylated RNAs.^[49] In contemporary full-length cDNA library construction, template-switching reverse transcription (TSRT) integrates second-strand initiation during or immediately after first-strand synthesis; a template-switching oligonucleotide (TSO) with degenerate nucleotides anneals to the 3' overhang created by terminal transferase activity of certain reverse transcriptases (e.g., M-MuLV variants), enabling primer extension for the second strand without RNase H digestion.^[50] This method, commercialized in systems like SMART cDNA, preserves 5' ends and facilitates unbiased amplification of low-abundance transcripts.^[51] Amplification techniques expand ds cDNA for analysis, often via polymerase chain reaction (PCR) following second-strand completion. In traditional workflows, blunt-end ds cDNA is ligated to adapters or vectors before PCR using universal primers flanking the insert, as in 5'/3' rapid amplification of cDNA ends (RACE) protocols.^[52] For quantitative reverse transcription PCR (qRT-PCR), first-strand cDNA is frequently used directly as a template, with PCR cycles synthesizing the second strand de novo using gene-specific primers, bypassing dedicated second-strand synthesis to minimize bias and artifacts from low-input RNA.^[53] TSRT-based systems amplify via PCR with primers targeting the TSO and poly(A) adapter, enabling exponential increase (up to 10^6-fold) while maintaining representation of transcript abundance.^[51] Commercial kits, such as those employing dUTP incorporation for strand-specificity, further refine amplification by allowing selective degradation of unwanted strands post-PCR.^[54] These techniques prioritize fidelity and coverage, though challenges like primer dimers and GC bias necessitate optimized cycling conditions (e.g., 94–98°C denaturation, 50–60°C annealing).^[55]

Applications in Research and Biotechnology

Gene Cloning and Expression Studies

Complementary DNA (cDNA) facilitates gene cloning by providing intron-free coding sequences derived from mature mRNA, enabling the isolation and amplification of eukaryotic genes that would otherwise be interrupted by non-coding introns in genomic DNA.^[56] This approach is particularly valuable for cloning expressed genes, as cDNA represents only the transcribed and processed portions of the genome, simplifying downstream manipulation and expression in heterologous systems.^[57] cDNA libraries, collections of cloned cDNA fragments inserted into vectors such as plasmids or bacteriophages, serve as primary resources for gene cloning.^[58] These libraries are constructed by reverse transcribing polyadenylated mRNA from specific tissues or cell types, followed by ligation of double-stranded cDNA into vectors and transformation into host cells like Escherichia coli. Screening methods, including hybridization with oligonucleotide probes complementary to known sequences or functional assays for phenotypic complementation, allow identification of target clones.^[59] For instance, functional cDNA expression cloning identifies full-length cDNAs based on their ability to confer selectable phenotypes, such as enzyme activity or resistance, upon expression in target cells.^[60] In expression studies, cloned cDNA inserts are placed under control of strong promoters in expression vectors to drive recombinant protein production.^[61] Since cDNA lacks native eukaryotic regulatory elements like introns and enhancers, vectors supply prokaryotic or eukaryotic promoters, ribosome-binding sites, and terminators tailored to the host—such as T7 promoters for bacterial systems or CMV promoters for mammalian cells.^[61] This enables high-yield protein expression; for example, cDNA-derived open reading frames (ORFs) are routinely used to produce fusion proteins with affinity tags like His-tags for purification and analysis.^[62] Applications include characterizing protein function, structure determination via X-ray crystallography, and generating antigens for diagnostics, with yields often reaching milligrams per liter in optimized bacterial or yeast systems.^[63] Challenges in expression, such as codon bias or post-translational modifications, are addressed by codon-optimization of cDNA sequences or selection of eukaryotic hosts like Pichia pastoris.^[64] Over the past three decades, advancements in cDNA cloning have expanded its utility in expression studies, including the creation of comprehensive libraries from human tissues for proteome-wide analysis.^[65] These tools have enabled the recombinant expression of thousands of human proteins, supporting functional genomics and therapeutic protein development, though success rates vary due to factors like mRNA abundance and secondary structure during synthesis.^[66]

Gene Expression Profiling and Diagnostics

Complementary DNA (cDNA) synthesized from messenger RNA (mRNA) serves as a stable intermediate for gene expression profiling by converting transient RNA transcripts into durable DNA copies amenable to amplification and hybridization techniques.^[67] In cDNA microarray analysis, RNA from a sample is reverse-transcribed into labeled cDNA targets, which are then hybridized to arrays containing thousands of immobilized cDNA probes derived from known genes; differential fluorescence intensities quantify relative expression levels across the transcriptome.^[67] This method, pioneered in the early 1990s with initial radioactive labeling approaches, enables simultaneous monitoring of thousands of genes, facilitating the identification of expression patterns associated with cellular states or perturbations.^[68] In diagnostics, cDNA microarrays provide expression profiles that distinguish pathological from normal tissues, aiding in disease classification and biomarker discovery.^[69] For instance, in oncology, they have been applied to profile gene expression in melanoma cell lines, revealing tumorigenic signatures through comparisons of normal and malignant samples as early as 1996.^[70] Clinical applications include screening for differentially expressed genes to identify novel therapeutic targets or prognostic markers, such as in breast cancer subtyping where specific expression patterns correlate with treatment response.^[69] Microarrays also support infectious disease diagnostics by detecting pathogen-specific transcripts or host responses, though their use has been supplemented by higher-resolution methods like RNA sequencing, which similarly relies on cDNA synthesis for library preparation.^[71] Validation of microarray findings often employs reverse transcriptase polymerase chain reaction (RT-PCR) on cDNA to confirm expression changes, ensuring reliability in diagnostic contexts.^[72] Advances in cDNA-based profiling have improved diagnostic precision, with targeted amplification methods like cDNA single-molecule molecular inversion probes enabling multiplexed quantification of low-abundance transcripts for applications in cancer biomarker detection.^[73] Despite limitations such as probe cross-hybridization, these techniques have informed personalized medicine by linking expression profiles to clinical outcomes, as seen in regulatory perspectives on microarray use for drug selection and therapy monitoring.^[74] Overall, cDNA's role underscores its utility in bridging RNA dynamics to actionable diagnostic insights grounded in empirical expression data.

Therapeutic and Drug Development Uses

Complementary DNA (cDNA) enables the production of recombinant therapeutic proteins by providing intron-free coding sequences that can be cloned into bacterial, yeast, or mammalian expression systems for large-scale manufacturing. This approach has been fundamental to developing biologics such as interferons, erythropoietin, and factor VIII, which are expressed from cDNA-derived genes to treat conditions like anemia, hemophilia, and hepatitis.^[75] For example, recombinant human erythropoietin, approved by the FDA in 1989, was produced using cDNA cloned into Chinese hamster ovary cells, revolutionizing treatment for chronic kidney disease-related anemia.^[76] In gene therapy, cDNA serves as the transgene payload in viral vectors to restore functional protein expression in genetic disorders. Gamma-retroviral vectors carrying IL2RG cDNA successfully treated severe combined immunodeficiency (SCID-X1) in early trials starting in 2000, achieving long-term immune reconstitution in patients.^[77] Similarly, Strimvelis, approved by the European Medicines Agency in 2016, delivers ADA cDNA via a gamma-retroviral vector to hematopoietic stem cells for adenosine deaminase deficiency-SCID, offering a one-time curative option.^[78] Zynteglo, approved in the European Union in 2019 and later by the FDA, uses a lentiviral vector with β-globin cDNA to treat transfusion-dependent β-thalassemia, enabling sustained hemoglobin production in treated patients.^[76] cDNA libraries and expression profiling support drug development by identifying novel targets and elucidating mechanisms of action or resistance. High-throughput screening of cDNA libraries has facilitated the discovery of tumor antigens for targeted therapies and vaccines, as demonstrated in phage display systems constructed from patient-derived cDNA.^[79] In pharmacogenomics, cDNA microarray-based gene expression databases have been applied to cancer pharmacology, correlating expression patterns with drug sensitivity to prioritize candidates, such as in NCI-60 cell line panels analyzed since 1999.^[80] Additionally, cDNA-derived sequences provide templates for in vitro transcription in developing mRNA therapeutics, including vaccines, where the coding region is amplified from cDNA for synthetic mRNA production.^[81]

Natural Biological Roles

In Retroviruses

In retroviruses, complementary DNA (cDNA) serves as the essential intermediate in converting the single-stranded positive-sense RNA genome into a double-stranded DNA form capable of integrating into the host cell's genome, thereby establishing persistent infection. This reverse transcription process occurs in the cytoplasm shortly after viral entry and is catalyzed by the virally encoded reverse transcriptase (RT) enzyme, which possesses both RNA-dependent DNA polymerase and RNase H activities.^[82] The resulting cDNA, initially single-stranded and complementary to the RNA template (the minus strand), undergoes further synthesis to form a linear double-stranded provirus.^[83] Reverse transcription begins when a host transfer RNA (tRNA) primer binds to the primer binding site (PBS) adjacent to the 5' unique region (U5) of the viral RNA genome, supplying the 3'-hydroxyl group required for nucleotide extension. RT then polymerizes deoxyribonucleotides along the RNA template, synthesizing the minus-strand strong-stop DNA (approximately 100-200 nucleotides) up to the 5' cap of the RNA. RNase H activity simultaneously degrades the RNA in the RNA-DNA hybrid, except for resistant segments like the polypurine tract (PPT), which primes plus-strand synthesis.^[84] A critical first strand transfer follows, where the newly synthesized minus-strand DNA anneals to the complementary repeat (R) region at the 3' end of the RNA genome via homologous base pairing, allowing extension to copy the full-length template.^[82] The process concludes with plus-strand synthesis initiating from the PPT primer, RNase H-mediated removal of the tRNA primer, a second strand transfer aligning the plus-strand U3 region with the minus-strand counterpart, and completion of both strands to yield full-length double-stranded cDNA flanked by long terminal repeats (LTRs). This LTR-capped cDNA is substrate for the viral integrase enzyme, which catalyzes its insertion into host chromatin as a provirus, from which viral genes are transcribed by host machinery.^[83] The fidelity of cDNA synthesis is low due to RT's error-prone nature, contributing to high mutation rates (approximately 10^{-4} to 10^{-5} errors per nucleotide per replication cycle) that enable viral evasion of host defenses and antiviral drugs.^[82] In human immunodeficiency virus type 1 (HIV-1), for instance, reverse transcription completes within 1-2 hours post-entry, with partial cDNA intermediates detectable if blocked by inhibitors like non-nucleoside RT inhibitors.^[85]

In Retrotransposons and Host Genomes

Retrotransposons, a class of transposable elements, replicate and propagate within host genomes through an RNA-mediated mechanism that centrally involves the synthesis of complementary DNA (cDNA). Unlike DNA transposons, which excise and reintegrate directly, retrotransposons are first transcribed into RNA by host RNA polymerases, serving dual roles as mRNA for protein translation (including reverse transcriptase) and as the template for cDNA production.^[86] Reverse transcription converts this single-stranded RNA into double-stranded cDNA, typically in the cytoplasm or nucleus depending on the element type, enabling retrotransposition by inserting the cDNA copy at new genomic loci.^[87] This process amplifies retrotransposon sequences, with long terminal repeat (LTR) retrotransposons employing an integrase enzyme analogous to retroviruses to catalyze cDNA integration, while non-LTR retrotransposons like LINE-1 utilize target-primed reverse transcription (TPRT), where the 3' hydroxyl of a cleaved target DNA site primes cDNA synthesis directly on the chromosome.^[88]^[89] Integration of retrotransposon-derived cDNA profoundly shapes host genomes, contributing to structural variation and functional evolution but also posing mutagenic risks. In eukaryotes, retrotransposons account for substantial portions of genomic DNA; for instance, they comprise over 40% of the human genome, with LINE-1 elements alone occupying about 17%.^[90] Successful cDNA insertion relies on host cofactors, including chromatin remodelers and DNA repair proteins, which facilitate access to integration hotspots such as gene-rich regions or heterochromatin boundaries, as observed in yeast Ty1 elements targeting upstream of tRNA genes.^[91] ^[92] While parasitic in amplifying their own copies, retrotransposons provide hosts with genetic raw material: cDNA insertions can donate exons, create alternative promoters, or rearrange host genes, influencing expression in contexts like oocyte development where they regulate early embryonic transcripts.^[93] However, erroneous integrations disrupt genes, promote genomic instability, and contribute to diseases including cancer via insertional mutagenesis.^[94] Certain retrotransposon families exhibit specialized cDNA handling that modulates host interactions. DIRS-like elements, for example, generate linear single-stranded cDNA intermediates rather than conventional double-stranded forms, which are then circularized and integrated via tyrosine recombinase, bypassing typical integrase dependency and potentially evading host silencing mechanisms.^[86] Non-integrase pathways also exist, as demonstrated in some LTR elements where cDNA recombines with homologous sequences independently of integrase, fostering genetic diversity through shuffling.^[95] These dynamics underscore a bidirectional relationship: hosts evolve suppressors like piRNAs and DNA methylation to curtail retrotransposition, yet tolerate or co-opt cDNA-derived sequences for adaptive traits, such as telomerase-related reverse transcription in maintaining chromosome ends.^[96] Empirical studies, including genetic screens in model organisms, reveal conserved host factors across species that either promote or restrict cDNA integration, highlighting the evolutionary arms race between retrotransposons and genomes.^[97]

Advancements and Challenges

Recent Technological Improvements

Recent advancements in complementary DNA (cDNA) synthesis have focused on enhancing reverse transcriptase (RT) processivity, fidelity, and adaptability to low-input samples and challenging RNA templates, particularly for integration with next-generation sequencing (NGS) workflows. Engineered RT variants, such as those derived from group II introns, enable ultraprocessive end-to-end RNA-to-cDNA conversion in a single enzymatic pass at ambient temperatures, overcoming limitations of traditional retroviral RTs in handling structured, long, or repetitive sequences.^[98] This approach improves transcript coverage and detection of rare isoforms or long noncoding RNAs, as demonstrated in commercial kits launched in 2025.^[98] Modifications to template-switching mechanisms have boosted sensitivity and coverage in high-throughput RNA analysis. A 2025 comparative study optimized template-switching cDNA synthesis using oligo(dT)23-VN primers combined with random hexamers, achieving up to 2.2-fold higher relative read abundance and 85.7% genome coverage for poly-A-tailed viral RNAs in complex plant matrices via Nanopore and Illumina platforms.^[99] These refinements reduce bias against full-length transcripts and enhance multiplex detection of latent viruses, outperforming anchored random priming in quarantine diagnostics.^[99] For long-read sequencing, the ordered two-template relay (OTTR) method saw iterative improvements in early 2025, incorporating Bombyx mori R2 RT mutants (e.g., W403A/F753A) and DNA-only 3' adapters capped with dideoxycytidine. These changes yielded 84% 3' end precision, coefficient of variation (CV) of 0.57–0.65 for bias reduction, and compatibility with 2.8 pg RNA inputs while minimizing contaminants below 10% at 3 pg.^[100] Biotinylated dideoxyadenosine labeling further streamlined gel-free duplex enrichment, facilitating low-bias libraries for noncoding RNA profiling.^[100] Automation and enzyme fidelity enhancements have also addressed error rates in RT-dependent DNA synthesis, with NGS-based assays quantifying reduced mutation frequencies in advanced RTs for precise gene expression studies.^[101] These developments collectively lower technical hurdles in single-cell and spatial transcriptomics, where full-length cDNA amplification via RTs with terminal transferase activity captures diverse transcript populations more comprehensively.^[102]

Limitations and Technical Hurdles

One primary technical hurdle in cDNA synthesis is the inherent error rate of reverse transcriptase enzymes, which lack 3'-5' exonuclease proofreading activity, leading to frequent nucleotide misincorporations during RNA-to-DNA conversion.^[103] Error rates can reach approximately 1 in 10,000 to 1 in 100,000 bases, depending on the enzyme variant, resulting in sequence inaccuracies that propagate into downstream applications like sequencing or cloning.^[104] High-fidelity engineered reverse transcriptases mitigate this but do not eliminate it entirely, and validation via sequencing is often required.^[105] Biases introduced during reverse transcription further distort the cDNA pool relative to the original mRNA transcriptome, including preferential amplification of shorter or more stable transcripts and underrepresentation of those with complex secondary structures.^[106] For instance, 3'-end bias arises from oligo(dT) priming strategies, which favor polyadenylated tails and overlook non-poly(A) RNAs or internal sequences, compromising comprehensive gene expression profiling.^[107] Ligation steps in library construction exacerbate this through sequence- or GC-content-dependent inefficiencies, necessitating bias-correction algorithms in data analysis.^[108] Achieving full-length cDNA remains challenging due to the limited processivity of reverse transcriptases and mRNA degradation, often yielding truncated products that miss 5'-ends critical for promoter studies or complete coding sequences.^[109] RNA quality is paramount; contaminants like salts, phenols, or genomic DNA inhibit the reaction or introduce artifacts, requiring stringent purification protocols such as DNase treatment.^[36]^[110] Additionally, spurious second-strand synthesis by some reverse transcriptases generates aberrant DNA products, complicating single-molecule analyses.^[111] In cDNA library construction, these issues compound with chimeric clones and size biases from random priming or fragmentation, reducing library diversity and representational accuracy compared to genomic libraries, which inherently lack introns and regulatory elements absent in mature mRNA.^[58] Scaling for high-throughput applications demands optimized conditions to minimize inter-sample variability, yet enzyme- and protocol-specific biases persist, as evidenced by comparative studies showing up to 2-fold distortions in transcript abundance.^[112] Ongoing advancements, such as template-switching methods, address some hurdles but introduce new ones like adapter dimer formation.^[113]

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Reverse transcriptases are commonly used to produce complementary DNA (cDNA) libraries from various expressed mRNAs and are also used to quantify the level of ...<|separator|>
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Exploring cDNA: Introduction, Synthesis, gDNA Comparison, and ...
Apr 26, 2025 · Reverse transcription takes mRNA as a template, and single-stranded DNA complementary to mRNA is synthesized by reverse transcriptase, and then ...
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26: Reverse Transcription PCR - Biology LibreTexts
Aug 22, 2024 · In eukaryotes, this means that a cDNA differs substantially from genomic DNA, since cDNA lacks introns. To amplify a particular RNA sequence ...
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Understanding a Genome Sequence - NCBI - NIH
Comparing a cDNA sequence with a genomic DNA sequence therefore delineates the position of the relevant gene and reveals the exon-intron boundaries. In order ...
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cDNA vs. gDNA: Structure, Function, Biotech Apps, and Why ...
Apr 26, 2025 · Researchers use reverse transcriptase, using mRNA as template, and synthesize complementary DNA chain, namely cDNA, according to the principle ...
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Half a century of the reverse transcriptase—happy birthday!
Jan 11, 2021 · The reverse transcriptase (RT) was discovered 50 years ago by Howard M. Temin, Satoshi Mitzutani [1], and David Baltimore [2].
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50th anniversary of the discovery of reverse transcriptase - PMC
Jan 15, 2021 · The simultaneous discovery in 1970 of reverse transcriptase in virions of retroviruses by Howard Temin and David Baltimore was perhaps the most dramatic ...
[22]
Reversal of Misfortune | Science | AAAS
Baltimore and oncologist Howard Temin independently discovered the enzyme, later called reverse transcriptase, that enables so-called retroviruses—including ...
[23]
Destroying Dogma: the Discovery of Reverse Transcriptase
So Baltimore and his colleagues searched for an enzyme that could make DNA from RNA. Looking first in Rous sarcoma virus (RSV) and then in Rauscher mouse ...
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The 50th anniversary of reverse transcriptase—and its ironic legacy ...
Jun 11, 2020 · On June 27, 1970, back-to-back papers were published in Nature presenting evidence that two oncogenic viruses contain an enzymatic activity that can synthesize ...Howard Temin · The Provirus Idea · Those Puzzling Rna VirusesMissing: date | Show results with:date
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The Discovery of Reverse Transcriptase - PubMed - NIH
In 1970, David Baltimore and Howard Temin independently discovered reverse transcriptase in retroviruses, revolutionizing molecular biology.
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The Nobel Prize in Physiology or Medicine 1975 - Press release
Karolinska institutet has decided to award the Nobel Prize in Physiology or Medicine for 1975 jointly to David Baltimore, Renato Dulbecco and Howard Temin.
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[PDF] THE DISCOVERY OF REVERSE TRANSCRIPTASE - ICB-USP
Temin and Baltimore were led inde- pendently by different key deductions to the discovery of reverse transcriptase. Temin firmly believed the activity.
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Reverse Transcriptases: From Discovery and Applications to ...
Nov 10, 2022 · They thus catalyze the reverse of transcription. Although discovered in 1970, reverse transcriptases are still of great interest and are ...
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[PDF] Reverse Transcriptase and cDNA Synthesis
This article presents an overview of reverse transcriptase from a historical viewpoint and describes how this enzyme is involved in retroviral replication ...<|control11|><|separator|>
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Reverse Transcription: An In-Depth Exploration of Structure ...
Apr 26, 2025 · In the process of reverse transcription, RNA-DNA hybrids will be formed with the synthesis of the first strand of cDNA. The RNase H activity of ...
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Preparation of cDNA libraries for high-throughput RNA sequencing ...
2. First strand cDNA synthesis. Mix 10 μl of 5′-Adaptor-ligated material (see Note 11) with 0.3 μl of RT primer (100 pmol/μl). As with the 5′ adaptor, the ...
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An Overview on RNA Isolation and Complementary DNA (cDNA ...
Phenol-chloroform extraction: This is one of the oldest and most reliable methods. It involves the use of phenol and chloroform to separate RNA from proteins ...
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Comparison of Four Methods of RNA Extraction and cDNA ... - NIH
Jul 6, 2023 · RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes.<|separator|>
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[PDF] A quick and cost-effective method for DNA-free total RNA isolation ...
Mar 23, 2023 · Here, we developed an optimized in-house protocol for isolating high-quality. RNA free of genomic DNA contamination using magnetic silica beads.<|separator|>
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Overview of cDNA Synthesis - CD Genomics
cDNA synthesis, achieved through the process of reverse transcription, involves transcribing RNA into DNA, thereby serving as a vital amendment and ...
[37]
mRNA Extraction and Reverse Transcription-PCR Protocol for ...
The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR ...
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M-MuLV reverse transcriptase: Selected properties and improved ...
Nov 22, 2021 · Thus, reverse transcriptases were proved to have three major enzymatic activities: RNA-dependent DNA synthesis, DNA-dependent DNA synthesis, ...
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High temperature cDNA synthesis by AMV reverse transcriptase ...
AMV-RT can synthesize cDNA at 70°C, which improves PCR specificity. This high temperature property raises questions about enzyme evolution.
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Reverse Transcription Reaction Setup | Thermo Fisher Scientific - US
Sep 30, 2015 · Primer annealing, DNA polymerization, and enzyme deactivation (Figure 6) are the three main steps of reverse transcription. The temperature and ...RNA template preparation · Genomic DNA removal · Primer selection
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Thermostable group II intron reverse transcriptase fusion ... - PubMed
Thermostable group II intron RT fusion proteins have higher processivity and fidelity than retroviral RTs, and are used in cDNA synthesis and RNA sequencing. ...
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A Novel Thermostable and Processive Reverse Transcriptase ... - NIH
RTs from thermophilic bacteria are thermostable enzymes that allow cDNA synthesis to be performed at temperatures around and above 50–60 °C. Higher reaction ...
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Shedding light: The importance of reverse transcription efficiency ...
Feb 12, 2019 · The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ...
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Discovery and evolution of RNA and XNA reverse transcriptase ...
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[PDF] Manual: Just cDNA Double-Stranded cDNA Synthesis Kit
During second-strand synthesis, RNase H nicks the RNA bound to the first- strand cDNA to produce a multitude of fragments, which serve as primers for DNA ...<|separator|>
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second strand cDNA synth without primer? - Molecular Biology
Feb 12, 2008 · Treatment with the RNAse H during the second step, cuts up the RNA, leaving small fragment bound to the DNA strand. These bits of of RNA serves ...
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Efficient second-strand cDNA synthesis using T7 DNA polymerase
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3´ RACE System for Rapid Amplification of cDNA Ends
Overview · The 3´ RACE procedure is summarized as follows. First strand cDNA synthesis is initiated at the poly(A) tail of mRNA using the adapter primer (AP).
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Is it necessary to synthesis second strand cDNA to amplify the gene ...
Nov 8, 2017 · In many cases, however, for PCR-based techniques, synthesizing the second strand is indeed a necessary step. Cite. 5 Recommendations. Martin ...Which is the best strategy to synthetize the cDNA second strand for ...Why degrade my cDNA during the second strand synthesis reaction?More results from www.researchgate.net
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Targeted rapid amplification of cDNA ends (T-RACE)—an improved ...
Sep 15, 2010 · Second-strand synthesis using a dUTP containing nucleotide mix is performed through PCR using primers (which also contain dUTP) complementary to ...
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[PDF] Second Strand cDNA Synthesis Kit | Thermo Fisher Scientific
May 21, 2020 · Second strand cDNA is generated using the first strand cDNA as a template. The Second Strand cDNA Synthesis Kit uses nick translational.
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cDNA cloning - www-users
We will begin by describing how a cDNA for a known protein can be isolated using amino acid sequence information, which, historically, was the first way that a ...Missing: history | Show results with:history
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Reverse Transcription Applications | Thermo Fisher Scientific - US
The cDNA library clones are used in the characterization of novel RNA transcripts, determination of gene sequences, and expression of recombinant proteins.
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cDNA Library Overview and Applications - Creative Biogene
Gene cloning: cDNA libraries are extensively used in gene cloning, which aids in the production of large quantities of proteins and diagnostic reagents.
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The Biotechnology Revolution: PCR and Cloning Expressed Genes
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Functional cDNA expression cloning: Pushing it to the limit - PMC
Step 1 is cDNA synthesis with the vector primer as a primer for reverse transcription with AMV reverse transcriptase. Step 2 is oligo(dC) tailing at the 3′-ends ...
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Why do we need Recombinant Proteins? - Enzo Biochem
Jan 26, 2023 · Because the cDNA lacks regulatory regions, the expression vectors provide promoter, ribosome-binding site, and terminator sequences.<|separator|>
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A catalog of human cDNA expression clones and its application to ...
Aug 17, 2004 · For expression of cDNA inserts as His-tag fusion proteins, the respective cDNA insert has to be cloned in-frame to the vector-encoded start ...
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cDNA & ORF clones - Gene overexpression - Horizon Discovery
cDNA and ORF clones provide a quick and accessible method for obtaining gene content for many in vitro and cell culture overexpression applications.
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Recombinant Protein & cDNA - | Bioclone
Codon-optimized cDNA & Recombinant Protein. The number of recombinant proteins/DNA used for academic research and therapeutic applications has increased
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cDNA Cloning - an overview | ScienceDirect Topics
Looking back at the development of cDNA cloning technologies over the past 30 years, there is a strong basis for developing novel approaches to the cloning and ...Missing: history | Show results with:history
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cDNA cloning, recombinant expression and characterization of ...
These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated ...
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High throughput gene expression profiling: a molecular approach to ...
Basically, thousands of cDNA clones are individually amplified by PCR and printed on glass slides using a robotic arrayer to produce microarrays of DNA probes.
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Historical Background and Anticipated Developments - PubMed
In fact, the first article describing the application of cDNA arrays to expression analysis was published in 1992, relied on radioactive labeling, and was a ...Missing: techniques | Show results with:techniques
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Clinical application of cDNA microarrays in oncology - PubMed
It may be used to screen for individual genes that are differentially expressed between normal and diseased tissues in the hope of finding novel targets for ...
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Use of a cDNA microarray to analyse gene expression patterns in ...
Dec 1, 1996 · Microarrays of cDNA provide a powerful tool for studying these complex phenomena6–8. The tumorigenic properties of a human melanoma cell line, ...
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DNA microarray technique for infectious disease diagnostics. | PLMI
Nov 10, 2023 · DNA microarray is a novel and advanced diagnostic test for infectious disease that is designed for specific identification of a wide variety of organisms ...
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Gene Expression Profile Analysis by DNA Microarrays - JAMA Network
Reverse transcriptase polymerase chain reaction (RT-PCR) is a method often used to verify microarray data. Although RT-PCR is not well suited to quantitation, ...
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Quantification of differential gene expression by multiplexed ... - Nature
May 5, 2017 · cDNA-smMIPs have the potential to address an important practical need in gene expression studies as a method that can measure expression of a ...
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[PDF] Medical applications of microarray technologies: a regulatory ...
Microarrays may be used for drug selection, disease diagnosis, and monitoring therapeutic interventions. They are also used for genome-wide genotyping and ...
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Recombinant Protein - an overview | ScienceDirect Topics
Recombinant human insulin was the first of these biotechnologically manufactured products that was approved by the US Food and Drug Administration (FDA) in 1982 ...Recombinant Proteins · End-Of-Chapter Questions · Translational Value Of...<|control11|><|separator|>
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A primer to gene therapy: Progress, prospects, and problems - PMC
Genetic therapies based on gene addition have witnessed a variety of clinical successes and the first therapeutic products have been approved for clinical use.
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Preparation of Phage Display cDNA Libraries for Identifying ... - NIH
Nov 29, 2024 · This review examines the discovery of tumour antigens using phage display technology, emphasising the construction of cDNA libraries, their subsequent display ...
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A cDNA microarray gene expression database for cancer drug ...
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mRNA-based therapeutics — developing a new class of drugs
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Overview of Reverse Transcription - Retroviruses - NCBI Bookshelf
Reverse transcription begins when the viral particle enters the cytoplasm of a target cell. The viral RNA genome enters the cytoplasm as part of a ...<|separator|>
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Retroviral reverse transcriptases - PMC - PubMed Central
Reverse transcription is a critical step in the life cycle of all retroviruses and related retrotransposons. This complex process is performed exclusively ...
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Understanding Retroviral Life Cycle and its Genomic RNA Packaging
These tRNAs provide the free 3′ hydroxyl group to initiate reverse transcription, a requirement of DNA synthesis as observed in all DNA polymerases. The 17 ...
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HIV-1 Reverse Transcription - PMC - PubMed Central
This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the ...
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DIRS retrotransposons amplify via linear, single-stranded cDNA ...
Mar 14, 2020 · This work provides the first experimental evidence to propose a general retrotransposition mechanism of the class of DIRS like tyrosine recombinase ...
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Retrotransposon life cycle and its impacts on cellular responses - PMC
In contrast, retrotransposons are transcribed by RNA polymerases and are converted into cDNA by reverse transcription in the nucleus or cytoplasm, depending on ...
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Reverse Transcription of Retroviruses and LTR Retrotransposons
This chapter will cover the process of reverse transcription, and the RTs that are involved in the replication of retroviruses and the related long terminal ...
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Structural mechanism of LINE-1 target-primed reverse transcription
Mar 6, 2025 · Retrotransposons are genetic sequences that can move through a host genome by means of an RNA intermediate (1). Long interspersed element–1 ( ...
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Ten things you should know about transposable elements
Nov 19, 2018 · TEs can donate their own genes to the host, but they can also add exons and rearrange and duplicate existing host genes. In humans, intronic Alu ...
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Host co-factors of the retrovirus-like transposon Ty1 | Mobile DNA
Aug 2, 2012 · This set of RHF genes includes several chromatin organization genes that have a potential role in the integration of Ty1 cDNA into the host ...
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Integration of the yeast retrotransposon Ty1 is targeted to regions ...
Retroviruses and their relatives, the LTR-containing retrotransposons, integrate newly replicated cDNA copies of their genomes into the genomes of their hosts ...
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Retrotransposons Regulate Host Genes in Mouse Oocytes and ...
TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos.
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The take and give between retrotransposable elements and their hosts
Retrotransposons mobilize via RNA intermediates and usually carry with them the agent of their mobility, reverse transcriptase.
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Identification of an integrase-independent pathway of ... - Science
Jun 29, 2022 · Retroviruses and long terminal repeat retrotransposons rely on integrase (IN) to insert their complementary DNA (cDNA) into the genome of ...
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Telomerase and retrotransposons: Reverse transcriptases ... - PNAS
Dec 20, 2011 · Formation of this “retrogenome” involves the synthesis of reverse transcripts and the subsequent or accompanying incorporation of cDNA into the ...
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Host factors that promote retrotransposon integration are similar in ...
Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is ...
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cDNA Synthesis and Amplification Kit for Enhanced Short-Read ...
Aug 28, 2025 · RNAConnect has launched its UltraMarathonRT cDNA Synthesis and Amplification Kit, designed to enhance short-read RNA sequencing workflows.
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A comparative template-switching cDNA approach for HTS-based ...
Jan 11, 2025 · Seven cDNA synthesis treatments were used to test simultaneously the relative abundance and coverage of the three most commonly latent RNA viruses found in ...
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Improved precision, sensitivity, and adaptability of ordered two ...
Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3′ and 5′ end-precision of sequence capture and minimized overall library ...
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Next-Generation Sequencing Methods to Determine the Accuracy of ...
In this review, we summarize the developments in this field that allowed the determination of RNA-dependent DNA synthesis error rates for different RTs.
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Advancements in single-cell RNA sequencing and spatial ...
Feb 4, 2025 · Full-length cDNA is synthesized from mRNA transcripts using reverse transcriptase with terminal transferase activity.
[102]
Reverse Transcription Technology - Roche Life Science
Reverse transcription is the process by which a reverse transcriptase enzyme mediates the creation of a DNA complement (complementary DNA or cDNA) from an RNA ...
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Reverse Transcription Errors and RNA–DNA Differences at Short ...
For example, if the RT error rate is high, then a large number of STRs in cDNA will vary in length, thereby reducing their mappability to the reference genome, ...
[104]
Reverse Transcription Troubleshooting | Thermo Fisher Scientific - US
Sequence error in cDNA ; Check the error rate or fidelity of the reverse transcriptase used. Also, verify the reliability of sequencing results from high-quality ...
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Artifacts and biases of the reverse transcription reaction in RNA ...
This review highlights how the synthesized cDNA pool might differ from the original RNA repertoire.
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Bias in RNA-seq Library Preparation: Current Challenges and ... - NIH
Apr 19, 2021 · Additionally, library construction methods are frequently biased, which is a main concern for RNA-seq data quality. Among them, PCR ...
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Evaluating bias-reducing protocols for RNA sequencing library ...
Jul 7, 2014 · The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library ...
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Full-length cDNAs: more than just reaching the ends
The isolation and mapping of cDNAs is an entrée to characterizing the information that is of significant biological relevance in the genome of an organism.
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[PDF] Critical factors for synthesis of cDNA for real-time PCR analysis
An important issue to consider is the choice of primers for the synthesis of cDNA from RNA. In addition, eliminating genomic DNA contamination in the RNA sample.
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Single-molecule direct RNA sequencing without cDNA synthesis - NIH
One limitation of cDNA-based approaches is the tendency of various reverse transcriptases (RTs) to generate spurious second-strand cDNA due to their DNA- ...
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Enzyme- and gene-specific biases in reverse transcription of RNA ...
May 18, 2020 · In general, across the entire dataset, a 2-fold increase of cDNA input into a qPCR reaction resulted in an average decrease of 0.99 in Cq value ...
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Comparative Evaluation of cDNA Library Construction Approaches ...
We compared two commercially available stranded cDNA library preparation kits, the NuGEN Ovation SoLo RNA-Seq System and the Illumina TruSeq Stranded Total RNA.2.1. Rna-Seq Experiment... · 3.2. Microbial Rna Analysis · 3.3. Human Rna AnalysisMissing: practices | Show results with:practices