15.5
相補的DNA
概要
メッセンジャーRNA(mRNA)に転写された遺伝子だけが活性化、すなわち発現します。したがって、科学者は細胞からmRNAを抽出し、さまざまな細胞や組織での遺伝子発現を調べることができます。科学者は、mRNAを逆転写して相補的なDNA（cDNA）に変換します。mRNAにはイントロン（ノンコーディ…
体内のほぼすべての細胞が同じDNAを持っています ですが 神経細胞や筋細胞などの様々な細胞の型は 異なる遺伝子を発現します これは特定の遺伝子だけが 各細胞のメッセンジャーRNA mRNA内に転写されるためです 研究室では mRNAはテンプレートとして 遺伝子の発現を研究するため 相補的DNA cDNAを合成して用いられます 共通する方法は細胞からRNAを抽出し その後 リボソームRNAや転移RNAなどの 別のRNAの型からmRNAを分離させます これは ビーズの列上でサンプルを ひと続きのチミンヌクレオチドと付着させて実行します これらのポリA鎖の結合 つまり アデニンヌクレオチドの一つの鎖は特に 真核細胞のmRNAにおける主要な3種類の末端上に存在します RNAの別の型は結合させず 洗い流します mRNAが分離した後 ポリTプライマーが ポリA鎖と結合し 逆転写酵素の起始点を提供し mRNAからcDNA内の一本鎖を転写します リボヌクレアーゼ酵素などの化学物質はこの時 RNAを分解させるために追加されます DNAポリメラーゼ酵素もさらに 相補鎖をcDNAと合成するため用いられ 結果的に二本鎖cDNAが生じます これは細菌性あるいはウイルスベクターに挿入され 分子生物学の研究に用いられます
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.