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Questions and Answers
What is the name of the enzyme that uses RNA as a template to synthesize a complementary DNA strand?
What is the name of the enzyme that uses RNA as a template to synthesize a complementary DNA strand?
Why is it essential to isolate RNA molecules from specific cell types?
Why is it essential to isolate RNA molecules from specific cell types?
What is the main reason why mRNA represents a very small percentage of the total RNAs?
What is the main reason why mRNA represents a very small percentage of the total RNAs?
Why is RNA chemically unstable compared to DNA?
Why is RNA chemically unstable compared to DNA?
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What is the purpose of enriching for mRNA in the total RNA population?
What is the purpose of enriching for mRNA in the total RNA population?
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What is the result of the process of transcription in gene expression?
What is the result of the process of transcription in gene expression?
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What is the purpose of breaking open the target cells in the first step of the procedure?
What is the purpose of breaking open the target cells in the first step of the procedure?
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Which of the following is NOT a type of RNA found in the total RNA fraction?
Which of the following is NOT a type of RNA found in the total RNA fraction?
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What is the primary function of the poly-A tail in mRNA molecules?
What is the primary function of the poly-A tail in mRNA molecules?
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What type of chromatography is used to purify mRNA molecules?
What type of chromatography is used to purify mRNA molecules?
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What is the function of the reverse transcriptase enzyme?
What is the function of the reverse transcriptase enzyme?
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What is the purpose of the oligo-dT primer in cDNA synthesis?
What is the purpose of the oligo-dT primer in cDNA synthesis?
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What is the result of the mRNA splicing process?
What is the result of the mRNA splicing process?
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What is the difference between cDNA and genomic DNA?
What is the difference between cDNA and genomic DNA?
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What is the purpose of the Rnase H enzyme in cDNA synthesis?
What is the purpose of the Rnase H enzyme in cDNA synthesis?
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What is the final product of the cDNA synthesis process?
What is the final product of the cDNA synthesis process?
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What is the primary difference between a genomic DNA sequence and a complementary DNA (cDNA) sequence?
What is the primary difference between a genomic DNA sequence and a complementary DNA (cDNA) sequence?
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What is the purpose of using a promoter element with a cDNA?
What is the purpose of using a promoter element with a cDNA?
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What is the primary advantage of using quantitative real-time PCR?
What is the primary advantage of using quantitative real-time PCR?
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What is the significance of a CT value of 40 or more in quantitative real-time PCR?
What is the significance of a CT value of 40 or more in quantitative real-time PCR?
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What is the primary difference between a cDNA library and a genomic library?
What is the primary difference between a cDNA library and a genomic library?
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What is the purpose of rinsing and washing the array after incubation?
What is the purpose of rinsing and washing the array after incubation?
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What is the purpose of hybridization in microarray analysis?
What is the purpose of hybridization in microarray analysis?
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What can be identified by comparing microarray data from different conditions?
What can be identified by comparing microarray data from different conditions?
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What is the purpose of microarray analysis in tumor profiling?
What is the purpose of microarray analysis in tumor profiling?
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What is the advantage of using cDNA libraries over genomic libraries?
What is the advantage of using cDNA libraries over genomic libraries?
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What is the purpose of using Taq polymerase in PCR?
What is the purpose of using Taq polymerase in PCR?
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What can be detected using microarrays with a mutant allele?
What can be detected using microarrays with a mutant allele?
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What is the purpose of using probes on a microarray?
What is the purpose of using probes on a microarray?
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What is a common application of microarray analysis?
What is a common application of microarray analysis?
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Study Notes
cDNA Synthesis
- In the 1970s, Howard Temin and David Baltimore discovered reverse transcriptase (RT), an enzyme that uses RNA as a template and synthesizes a complementary DNA strand from the mRNA template.
- The process of copying RNA into DNA was virtually unknown until the discovery of RT.
- To clone a gene, it is essential to start with cells that express the protein of interest, such as human pancreatic islet cells for cloning the insulin gene.
Isolation of RNA
- The target cells are harvested, broken open (lysed), and the RNA is separated from other cellular components.
- The total RNA fraction contains mRNA, tRNA, rRNA, and small nuclear and nucleolar RNAs; mRNA represents a small percentage of the total RNAs.
- Ribosomal RNAs are usually the most abundant RNAs in an active cell due to the need for large amounts of ribosomes for protein synthesis.
- RNA is chemically unstable compared to DNA and is rapidly degraded by ribonucleases (RNAse).
Purification of mRNA
- Scientists developed a sterile, one-step purification process to enrich for mRNA using the poly-A tail attached to almost all mRNA molecules.
- The poly-A tail is used as a tool to separate mRNA from other RNAs in the total RNA fraction using affinity chromatography.
Construction of cDNA Library
- The mRNA molecules are converted into complementary DNA using the reverse transcriptase enzyme.
- The single-stranded DNA made by reverse transcriptase is then converted into double-stranded DNA in preparation for ligation into vectors.
- The cDNA sequence is a copy of a fully processed and spliced mRNA, which means that the cDNA sequence contains only exons and lacks introns removed during mRNA splicing.
Characteristics of cDNA
- A complementary DNA (cDNA) contains just the sequences that represent a copy of a mature mRNA.
- If provided with a promoter element, the cDNA can be used to express the protein encoded in the exon sequences, avoiding the need to properly process or splice the RNA product in cells.
Quantitative Real-Time PCR
- Quantitative real-time PCR detects PCR product growth throughout the amplification process.
- It requires a minute amount of DNA, cDNA, primers, thermostable polymerase, and a fluorescent dye.
- The cycle threshold (CT) value is the cycle at which a significant increase in ΔRn is first detected and is used for quantitation.
Applications of cDNA Libraries and Microarrays
- Cell-specific gene expression: identify genes that are expressed in specific cells by analyzing cDNA levels.
- Gene regulation: compare microarray data to reveal genes that are induced under one set of conditions and repressed under another.
- Elucidation of metabolic pathways: genes that encode proteins that participate in a common metabolic pathway are often expressed in a parallel manner.
- Tumor profiling: subtyping tumors that are sometimes morphologically indistinguishable based on differences in their gene expression levels.
- Genetic variation: microarrays can be used to identify disease-causing alleles in humans and mutations that contribute to quantitative traits in plants and other species.
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Description
Learn about the discovery of reverse transcriptase, an enzyme that synthesizes DNA from RNA, and the importance of isolating RNA for gene cloning. Understand how this process is crucial for cloning genes like insulin.