15.5
Uzupełniające DNA
Przegląd
Tylko geny, które ulegają transkrypcji do informacyjnego RNA (mRNA), są aktywne lub ulegają ekspresji. Naukowcy mogą zatem wyodrębnić mRNA z…
Almost every cell in the body has the same DNA, but different cell types, such as neurons and muscle cells, express different genes because only certain genes are transcribed into messenger RNA, or mRNA, in each cell. In the laboratory, mRNAs can be used as a template to synthesize complementary DNA, cDNA, to study gene expression. A common method is to extract RNA from cells, then isolate the mRNA from other types of RNA, like ribosomal RNA or transfer RNA, by running the sample over a column of beads with stretches of thymine nucleotides attached.
These bind to the poly-A tail, a chain of adenine nucleotides specifically present on the 3-prime ends of eukaryotic mRNA. The other types of RNA do not bind and are washed away.
After the mRNA is isolated, a poly-T primer is bound to the poly-A tail, providing a starting point for reverse transcriptase enzymes to transcribe a single-stranded cDNA from the mRNA. Chemicals, such as RNase enzymes, are then added to degrade the RNA.
DNA polymerase enzymes are then used to synthesize a strand complementary to the cDNA, resulting in double-stranded cDNA, which can be inserted into a bacterial or viral vector and used in 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.