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Complementary DNA
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Complementary DNA
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.
In engineered forms, it often is a copy (replicate) of the naturally occurring DNA from any particular organism's natural genome; the organism's own mRNA was naturally transcribed from its DNA, and the cDNA is reverse transcribed from the mRNA, yielding a duplicate of the original DNA. Engineered cDNA is often used to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), or to sequence or quantify mRNA molecules using DNA based methods (qPCR, RNA-seq). cDNA that codes for a specific protein can be transferred to a recipient cell for expression as part of recombinant DNA, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.
In natural forms, cDNA is produced by retroviruses (such as HIV-1, HIV-2, simian immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates a provirus.
The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU).
Patentability of cDNA was a subject of a 2013 US Supreme Court decision in Association for Molecular Pathology v. Myriad Genetics, Inc. As a compromise, the Court declared, that exons-only cDNA is patent-eligible, whereas isolated sequences of naturally occurring DNA comprising introns are not.
RNA serves as a template for cDNA synthesis. In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into target genomic DNA. In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA is then synthesized through in vitro reverse transcription.
RNA is transcribed from genomic DNA in host cells and is extracted by first lysing cells then purifying RNA utilizing widely used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods. Extraction methods vary depending on the source material. For example, extracting RNA from plant tissue requires additional reagents, such as polyvinylpyrrolidone (PVP), to remove phenolic compounds, carbohydrates, and other compounds that will otherwise render RNA unusable. To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation. Importantly, RNA integrity is maintained by inactivating RNases with chaotropic agents such as guanidinium isothiocyanate, sodium dodecyl sulphate (SDS), phenol or chloroform. Total RNA is then separated from other cellular components and precipitated with alcohol. Various commercial kits exist for simple and rapid RNA extractions for specific applications. Additional bead-based methods can be used to isolate specific sub-types of RNA (e.g. mRNA and microRNA) based on size or unique RNA regions.
Using a reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced RNase H activity suited for transcription of longer RNAs. The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature). cDNA is commonly generated from mRNA for gene expression analyses such as RT-qPCR and RNA-seq. mRNA is selectively reverse transcribed using oligo-dT primers that are the reverse complement of the poly-adenylated tail on the 3' end of all mRNA. The oligo-dT primer anneals to the poly-adenylated tail of the mRNA to serve as a binding site for the reverse transcriptase to begin reverse transcription. An optimized mixture of oligo-dT and random hexamer primers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias. Ribosomal RNA may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some non-coding RNA.
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Complementary DNA
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.
In engineered forms, it often is a copy (replicate) of the naturally occurring DNA from any particular organism's natural genome; the organism's own mRNA was naturally transcribed from its DNA, and the cDNA is reverse transcribed from the mRNA, yielding a duplicate of the original DNA. Engineered cDNA is often used to express a specific protein in a cell that does not normally express that protein (i.e., heterologous expression), or to sequence or quantify mRNA molecules using DNA based methods (qPCR, RNA-seq). cDNA that codes for a specific protein can be transferred to a recipient cell for expression as part of recombinant DNA, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.
In natural forms, cDNA is produced by retroviruses (such as HIV-1, HIV-2, simian immunodeficiency virus, etc.) and then integrated into the host's genome, where it creates a provirus.
The term cDNA is also used, typically in a bioinformatics context, to refer to an mRNA transcript's sequence, expressed as DNA bases (deoxy-GCAT) rather than RNA bases (GCAU).
Patentability of cDNA was a subject of a 2013 US Supreme Court decision in Association for Molecular Pathology v. Myriad Genetics, Inc. As a compromise, the Court declared, that exons-only cDNA is patent-eligible, whereas isolated sequences of naturally occurring DNA comprising introns are not.
RNA serves as a template for cDNA synthesis. In cellular life, cDNA is generated by viruses and retrotransposons for integration of RNA into target genomic DNA. In molecular biology, RNA is purified from source material after genomic DNA, proteins and other cellular components are removed. cDNA is then synthesized through in vitro reverse transcription.
RNA is transcribed from genomic DNA in host cells and is extracted by first lysing cells then purifying RNA utilizing widely used methods such as phenol-chloroform, silica column, and bead-based RNA extraction methods. Extraction methods vary depending on the source material. For example, extracting RNA from plant tissue requires additional reagents, such as polyvinylpyrrolidone (PVP), to remove phenolic compounds, carbohydrates, and other compounds that will otherwise render RNA unusable. To remove DNA and proteins, enzymes such as DNase and Proteinase K are used for degradation. Importantly, RNA integrity is maintained by inactivating RNases with chaotropic agents such as guanidinium isothiocyanate, sodium dodecyl sulphate (SDS), phenol or chloroform. Total RNA is then separated from other cellular components and precipitated with alcohol. Various commercial kits exist for simple and rapid RNA extractions for specific applications. Additional bead-based methods can be used to isolate specific sub-types of RNA (e.g. mRNA and microRNA) based on size or unique RNA regions.
Using a reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced RNase H activity suited for transcription of longer RNAs. The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature). cDNA is commonly generated from mRNA for gene expression analyses such as RT-qPCR and RNA-seq. mRNA is selectively reverse transcribed using oligo-dT primers that are the reverse complement of the poly-adenylated tail on the 3' end of all mRNA. The oligo-dT primer anneals to the poly-adenylated tail of the mRNA to serve as a binding site for the reverse transcriptase to begin reverse transcription. An optimized mixture of oligo-dT and random hexamer primers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias. Ribosomal RNA may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some non-coding RNA.
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