Related Experiment Video
Updated: Jul 7, 2026
10:34
Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
S1 analysis of messenger RNA using single-stranded DNA probes
1Massachusetts General Hospital, Boston, Massachusetts, USA.
Current Protocols in Molecular Biology
|February 12, 2008
Summary
This method precisely maps RNA 5' ends using labeled oligonucleotides and S1 nuclease. It enables nucleotide-level determination of transcriptional start sites and quantitation of mRNA levels.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Oligonucleotide labeling is crucial for sensitive molecular detection.
- Accurate mapping of RNA 5' ends is essential for understanding gene regulation.
Purpose of the Study:
- To describe a method for precise mapping of RNA 5' ends.
- To enable nucleotide-level determination of transcriptional start sites.
- To facilitate quantitation of mRNA levels.
Main Methods:
- 5'-end labeling of oligonucleotides using kinase.
- Hybridization to a complementary template and extension with Klenow fragment.
- Restriction enzyme digestion, alkaline agarose gel electrophoresis, and S1 nuclease digestion.
- Denaturing polyacrylamide gel electrophoresis for fragment length determination.
Main Results:
- Precise mapping of RNA 5' ends to the nucleotide.
- Determination of transcriptional start sites.
- Quantitation of relative mRNA levels through probe excess hybridization.
Conclusions:
- This protocol provides a robust method for high-resolution mapping of RNA 5' ends.
- The technique is valuable for studying gene expression and regulation.
- It offers a means for accurate quantitation of mRNA abundance.
Related Concept Videos
02:57Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
03:21RNA-seq
RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
02:24Ribosome Profiling
Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...