mRNA and cDNA Synthesis in Eukaryotes and Bacteria

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Questions and Answers

What is the method of choice for high-throughput RNA expression analysis?

MPSS analysis

DNA microarray technology

cDNA sequence sampling

Microarray hybridization (correct)

What technique allows the detection of unlabeled cDNA sequences?

SAGE technique

Microarray hybridization

MPSS analysis

Gold nanoparticles technique (correct)

Which technology is reported to increase sensitivity by using gold nanoparticles rather than fluorescent tags?

MPSS analysis (correct)

SAGE

cDNA sequence sampling

DNA microarray technology

What type of elements are arranged as spots in a grid pattern on a DNA microarray?

DNA elements Signup and view all the answers

In DNA microarray technology, the arrayed targets are hybridized with what type of probe?

Complex probes with different sequences Signup and view all the answers

What technique has been applied in whole-transcriptome analysis in Arabidopsis?

MPSS analysis Signup and view all the answers

What is the role of the Bbv1 enzyme in the process described?

Chewing a small fragment from the cDNA Signup and view all the answers

What determines the resulting four-base overhang in the process?

The cDNA sequence Signup and view all the answers

What is the purpose of the adaptors in the process?

Annealing to cleaved cDNA Signup and view all the answers

How does the encoding adaptor contribute to the process?

Allowing repetition of the process Signup and view all the answers

What was used to determine the accuracy of the method described?

MPSS analysis on yeast cells Signup and view all the answers

How many MPSS signatures were obtained from induced THP-1 cells during expression analysis?

Over 1.5 million Signup and view all the answers

What is one key difference between eukaryotic mRNA and bacterial mRNA regarding polyadenylated tails?

Eukaryotic mRNA has a polyadenylated tail used for cDNA synthesis, while bacterial mRNA generally lacks this tail. Signup and view all the answers

Why is selective priming with polyadenylated tails used for cDNA synthesis in eukaryotes?

To generate a labeled probe devoid of rRNA and tRNA. Signup and view all the answers

Why did Richmond and colleagues use glass microarrays with a fluorescent probe in their study on E.coli transcriptional profiling?

To obtain more reliable and consistent results compared to nylon arrays with a radioactive probe. Signup and view all the answers

How was the effect of isoniazid on Mycobacterium tuberculosis identified by Wilson et al. in 1999?

By exposing the bacteria to isoniazid and analyzing induced genes with a DNA array. Signup and view all the answers

In bacterial gene expression studies, what has microarray hybridization been primarily used for?

To identify potential new drug targets. Signup and view all the answers

What was one significant finding when E.coli was analyzed after exposure to heat shock by Richmond and colleagues?

The presence of rRNA did not interfere with the sensitivity of hybridization. Signup and view all the answers

Study Notes

MPSS Analysis

MPSS technique allows detection of unlabeled cDNA sequences, increasing sensitivity with gold nanoparticles instead of fluorescent tags.
MPSS has been applied in several studies, including whole-transcriptome analysis in Arabidopsis and comparison of human cell lines.

DNA Microarray Technology

DNA microarray technology allows parallel analysis of thousands of genes on a miniature device.
Microarray hybridization is the method of choice for high-throughput RNA expression analysis.
DNA microarrays comprise a series of DNA elements arranged as spots (features) in a grid pattern on a miniature solid support.

MPSS Process

The initiating primer recognizes the DpnII overhang, and carries the recognition site for a type IIs enzyme, Bbv1.
The cleaved cDNA is annealed to a set of 16 encoded adaptors, each recognizing a specific 4-nt overhang.
Scanning of the bead after each round of hybridization reveals the 4-bp overhang, generating a sequence signature for each cDNA.

Accuracy and Applications

The accuracy of MPSS was determined by analyzing early and late log phase yeast cells, with over 90% of signatures matching sequences in public databases.
MPSS was used for expression analysis on human THP-1 cells, obtaining over 1.5 million MPSS signatures.

Eukaryotes and Bacteria

In eukaryotes, mRNA has a polyadenylated tail, allowing selective priming of first-strand cDNA synthesis and generating a labeled probe devoid of rRNA and tRNA.
Bacterial mRNA generally lacks a polyadenylate tail, so hybridization is carried out with total RNA.

Bacterial Expression Profiling

Genome-wide transcriptional profiling has been used to study E. coli response to heat shock and lactose analog IPTG.
Expression profiling has been used to identify potential new drug targets in bacteria, such as identifying genes involved in biochemical pathways.

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Description

Learn about the differences in mRNA structure between eukaryotes and bacteria, and how this impacts cDNA synthesis and hybridization. Explore the use of polyadenylated tails in eukaryotic mRNA and the genome-wide transcriptional profile of E. coli.