15.5
Complementary DNA
Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study ge…
Almost every cell in the body has the same DNA.
However, different cell types, such as neurons and muscle cells, express different sets of genes. These genes are transcribed into different messenger RNAs, or mRNAs, in each cell.
In the laboratory, mRNA can be used as a template to synthesize complementary DNA, or cDNA. Since cDNA is synthesized from cellular mRNA, it represents the genes expressed by the cell.
One common method begins with extracting RNA from cells.
Following cell lysis and removal of cellular debris, the mRNA is isolated from other types of RNA, such as ribosomal RNA and transfer RNA. This is commonly done by adding the sample to a column containing beads with short stretches of thymine nucleotides attached.
These thymine stretches bind to the poly-A tail, a chain of adenine nucleotides found at the 3-prime end of eukaryotic mRNA, through complementary base pairing.
Other types of RNA lack poly-A tails, so they do not bind to the column and are washed away.
An elution buffer with a low salt concentration is then added. This disrupts the interaction between the poly-A tail and the thymine stretches, releasing the bound mRNA from the beads.
After the mRNA is isolated, an oligo-dT primer binds to the poly-A tail and provides a starting point for reverse transcriptase enzymes to synthesize a single-stranded cDNA copy of the mRNA.
RNase enzymes are then added to degrade the RNA strand of the RNA-cDNA hybrid.
Next, DNA polymerase enzymes synthesize a complementary DNA strand, producing double-stranded cDNA.
This cDNA can then be inserted into plasmids or viral vectors for molecular biology research.
View the full transcript and gain access to JoVE Core videos
Q1: Why do scientists use complementary DNA instead of genomic DNA to study gene expression?
Complementary DNA (cDNA) lacks introns and regulatory sequences present in genomic DNA, allowing researchers to directly determine the amino acid sequence of encoded peptides. Since cDNA is synthesized from mRNA, it represents only the genes actively expressed in specific cells or tissues, making it ideal for studying gene expression patterns across different cell types.
Q2: How does the poly-A tail help isolate mRNA from other RNA types?
Mature eukaryotic mRNA has a poly-A tail—a string of adenine nucleotides—added to its 3' end, while transfer RNA and ribosomal RNA do not. Researchers attach thymine nucleotides (oligo-dTs) to a column or magnetic beads, which specifically base-pair with the poly-A tail. This allows mRNA to be captured while other RNA types are washed away.
Q3: What role does reverse transcriptase play in cDNA synthesis?
Reverse transcriptase is a DNA polymerase enzyme from retroviruses that synthesizes a single-stranded cDNA strand from mRNA. Since reverse transcriptase can only add nucleotides to the 3' end of a chain, a poly-T primer binds to the poly-A tail to provide a starting point for cDNA synthesis.
Q4: Why is the RNA degraded after the first cDNA strand is synthesized?
After reverse transcriptase creates single-stranded cDNA, the original mRNA template must be removed to allow DNA polymerase to synthesize the complementary second strand. RNA is degraded using alkali treatment or RNase enzymes, leaving the single-stranded cDNA intact for the next synthesis step.
Q5: How is double-stranded cDNA created from single-stranded cDNA?
DNA polymerase synthesizes a second DNA strand complementary to the first cDNA strand, often using the hairpin loop at the end of the first strand or a nicked piece of the mRNA as a primer. This produces double-stranded cDNA, which can be inserted into bacterial or viral vectors for molecular biology research.
Q6: What is a cDNA library and why would researchers construct one?
A cDNA library is a collection representing all the mRNAs expressed in specific cells or tissues of interest. Researchers construct cDNA libraries to comprehensively study gene expression patterns, identify active genes in particular cell types, and preserve genetic information for additional research and analysis.
Q7: How does different gene expression in neurons versus muscle cells relate to cDNA analysis?
Although neurons and muscle cells contain the same DNA, they express different genes because only certain genes are transcribed into mRNA in each cell type. By extracting and analyzing mRNA from these different cell types, researchers can use cDNA synthesis to study which genes are active and understand cell-type-specific gene expression patterns.