cDNA Synthesis and Isolation of RNA

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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?

DNA polymerase

Protease

RNA polymerase

Reverse transcriptase (correct)

Why is it essential to isolate RNA molecules from specific cell types?

To understand the mechanism of translation

To clone the gene of interest (correct)

To study the structure of RNA

To study the function of ribosomes

What is the main reason why mRNA represents a very small percentage of the total RNAs?

Because tRNA is difficult to isolate

Because mRNA is highly unstable

Because rRNA is highly abundant (correct)

Because snRNA is highly expressed

Why is RNA chemically unstable compared to DNA?

Because RNA is more prone to degradation by ribonucleases Signup and view all the answers

What is the purpose of enriching for mRNA in the total RNA population?

To clone the gene of interest Signup and view all the answers

What is the result of the process of transcription in gene expression?

DNA is used as a template to make mRNA Signup and view all the answers

What is the purpose of breaking open the target cells in the first step of the procedure?

To separate the RNA from other cellular components Signup and view all the answers

Which of the following is NOT a type of RNA found in the total RNA fraction?

DNA Signup and view all the answers

What is the primary function of the poly-A tail in mRNA molecules?

To distinguish mRNA from other types of RNA Signup and view all the answers

What type of chromatography is used to purify mRNA molecules?

Affinity chromatography Signup and view all the answers

What is the function of the reverse transcriptase enzyme?

To convert RNA to DNA Signup and view all the answers

What is the purpose of the oligo-dT primer in cDNA synthesis?

To base pair with the poly-A tail and serve as a primer for reverse transcriptase Signup and view all the answers

What is the result of the mRNA splicing process?

The removal of introns from mRNA Signup and view all the answers

What is the difference between cDNA and genomic DNA?

cDNA sequence contains only exons and lacks introns, while genomic DNA contains both exons and introns Signup and view all the answers

What is the purpose of the Rnase H enzyme in cDNA synthesis?

To remove the mRNA strand from the DNA:RNA heteroduplex Signup and view all the answers

What is the final product of the cDNA synthesis process?

A double-stranded cDNA molecule Signup and view all the answers

What is the primary difference between a genomic DNA sequence and a complementary DNA (cDNA) sequence?

The presence of introns in genomic DNA Signup and view all the answers

What is the purpose of using a promoter element with a cDNA?

To facilitate protein expression Signup and view all the answers

What is the primary advantage of using quantitative real-time PCR?

It collects data during the exponential phase Signup and view all the answers

What is the significance of a CT value of 40 or more in quantitative real-time PCR?

It means no amplification occurred Signup and view all the answers

What is the primary difference between a cDNA library and a genomic library?

The length of DNA fragments cloned in cDNA libraries Signup and view all the answers

What is the purpose of rinsing and washing the array after incubation?

To remove the unpaired DNA samples Signup and view all the answers

What is the purpose of hybridization in microarray analysis?

To wash the DNA sample over the microarray Signup and view all the answers

What can be identified by comparing microarray data from different conditions?

Genes that are induced under one condition and repressed under another Signup and view all the answers

What is the purpose of microarray analysis in tumor profiling?

To identify subtypes of tumors based on gene expression levels Signup and view all the answers

What is the advantage of using cDNA libraries over genomic libraries?

They exclude introns and other non-coding regions Signup and view all the answers

What is the purpose of using Taq polymerase in PCR?

To facilitate the amplification of the DNA sample Signup and view all the answers

What can be detected using microarrays with a mutant allele?

Disease-causing alleles in humans Signup and view all the answers

What is the purpose of using probes on a microarray?

To pair with the DNA or cDNA samples for analysis Signup and view all the answers

What is a common application of microarray analysis?

Elucidating metabolic pathways Signup and view all the answers

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.