4.7
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
View the full transcript and gain access to JoVE Core videos
Q1: What is complementary DNA and how is it made?
Complementary DNA (cDNA) is synthesized from a messenger RNA (mRNA) template using the enzyme reverse transcriptase. This process converts the genetic information stored in RNA back into DNA form, creating a stable copy that represents only the expressed genes from a specific cell or tissue at a particular time.
Q2: Why is complementary DNA useful in molecular biology research?
Complementary DNA is valuable because it represents only actively expressed genes, eliminating introns and non-coding sequences found in genomic DNA. This makes cDNA ideal for studying gene expression patterns, cloning genes, and creating cDNA libraries that reflect the functional genes present in specific cell types or developmental stages.
Q3: How does complementary DNA differ from genomic DNA?
Complementary DNA contains only exons and represents genes actively transcribed in a cell, while genomic DNA includes all DNA sequences, including introns and regulatory regions. cDNA is synthesized from mRNA and lacks introns, making it smaller and more suitable for expressing eukaryotic genes in prokaryotic systems.
Q4: What role does reverse transcriptase play in cDNA synthesis?
Reverse transcriptase is the key enzyme that catalyzes cDNA synthesis by reading an mRNA template and synthesizing a complementary DNA strand. This enzyme enables researchers to convert the temporary RNA message into stable DNA that can be amplified, cloned, and analyzed using standard molecular biology techniques.
Q5: How can complementary DNA be amplified and analyzed?
Complementary DNA can be amplified using PCR or rapid amplification of cDNA ends (RACE) techniques, then analyzed through methods like agarose gel electrophoresis for separation of DNA fragments or sequencing. These approaches allow researchers to identify specific genes and determine their expression levels in different tissues or conditions.
Q6: What are cDNA libraries and why are they important?
A cDNA library is a collection of cloned cDNA sequences representing all genes expressed in a particular cell type or tissue. These libraries are important for functional genomics research, allowing scientists to study gene expression patterns, identify tissue-specific genes, and isolate genes for further characterization and protein production.
Q7: How is complementary DNA used in gene expression studies?
Complementary DNA serves as the template for measuring gene expression through techniques like real-time RT-PCR, which quantifies mRNA levels. Since cDNA represents only expressed genes, it provides an accurate snapshot of which genes are active in specific cells, tissues, or disease states.