15.5
DNA Complementare
Panoramica
Solo i geni trascritti in RNA messaggero (mRNA) sono attivi o espressi. Gli scienziati possono, quindi, estrarre l'mRNA dalle cellule per s…
- [Narratore] Quasi tutte le cellule del corpo
hanno lo stesso DNA, ma diversi tipi
di cellule, come neuroni e cellule muscolari,
esprimono geni diversi perché solo determinati geni
sono trascritti in RNA messaggero, o mRNA, in ogni cellula.
In laboratorio gli mRNA sono utilizzati come modello
per sintetizzare l'DNA gratuito, cDNA,
per studiare l'espressione genica.
Un metodo comune è quello di estrarre l'RNA dalle cellule,
quindi isolare l'mRNA da altri tipi di RNA,
come l'RNA ribosomiale o l'RNA transfer,
eseguendo il campione su una colonna di perle
con tratti di nucleotidi di timina attaccati.
Questi si legano alla coda Poly (A),
una catena di nucleotidi adenina specificamente presente
sui tre estremi dell'mRNA eucariotico.
Gli altri tipi di RNA non si legano e vengono lavati via.
Dopo che l'mRNA è stato isolato, un primer Poly (T)
è legato alla coda Poly (A), fornendo un punto di partenza
per gli enzimi della trascrittasi inversa
per trascrivere un singolo filamento in cDNA dall'mRNA.
Vengono aggiunti prodotti chimici come gli enzimi RNasi
per degradare l'RNA.
Vengono quindi utilizzati enzimi DNA polimerasi
sintetizzare un filo complementare al cDNA,
risultante in cDNA a doppio filamento,
che può essere inserito in un vettore batterico o virale
e utilizzato nella ricerca di biologia molecolare.
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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.