15.5
Komplementäre DNA
Überblick
Nur Gene, die in Boten-RNAs (mRNA) umgesetzt werden, sind aktiv oder werden exprimiert. Wissenschaftler können daher die mRNA aus Zellen ext…
Fast jede Zelle im Körper hat die gleiche DNA, aber verschiedene Zelltypen wie Neuronen und Muskelzellen exprimieren unterschiedliche Gene, da in jeder Zelle nur bestimmte Gene in Messenger-RNA oder mRNA transkribiert werden. Im Labor können mRNAs als Matrize zur Synthese komplementärer DNA (cDNA) zur Untersuchung der Genexpression verwendet werden. Eine übliche Methode besteht darin, RNA aus Zellen zu extrahieren und dann die mRNA aus anderen RNA-Typen wie ribosomaler RNA oder Transfer-RNA zu isolieren, indem die Probe über eine Kügelchensäule mit anhaftenden Thyminnukleotid-Abschnitten geführt wird.
Diese binden an den Poly(A)-Schwanz, eine Kette von Adeninnukleotiden, die spezifisch an den 3 primären Enden der eukaryotischen mRNA vorhanden sind. Die anderen RNA-Typen binden nicht und werden weggespült. Nachdem die mRNA isoliert wurde, wird ein Poly(T)-Primer an den Poly(A)-Schwanz gebunden, was einen Ausgangspunkt für reverse Transkriptaseenzyme darstellt, um einen Einzelstrang in cDNA aus der mRNA zu transkribieren.
Chemikalien wie RNase-Enzyme werden dann zugesetzt, um die RNA abzubauen. DNA-Polymeraseenzyme werden dann verwendet, um einen zur cDNA komplementären Strang zu synthetisieren, was zu doppelsträngiger cDNA führt, die in einen bakteriellen oder viralen Vektor inseriert und in der molekularbiologischen Forschung verwendet werden kann. cDNA - bakterieller Vektor
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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.