Related Experiment Video
Updated: Aug 9, 2026
12:24
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
Published on: July 2, 2010
Molecular cloning of sequence-specific DNA binding proteins using recognition site probes
H Singh1, R G Clerc, J H LeBowitz
1Massachusetts Institute of Technology.
Biotechniques
|March 1, 1989
Summary
Researchers can now isolate genes for DNA-binding proteins using a novel screening method. This technique utilizes recognition site DNA to identify transcriptional regulatory proteins, including those with helix-turn-helix, zinc finger, and leucine zipper motifs.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Identifying genes for sequence-specific DNA binding proteins is crucial for understanding gene regulation.
- Traditional methods for gene isolation can be laborious and time-consuming.
Purpose of the Study:
- To present a refined strategy for isolating genes encoding sequence-specific DNA binding proteins.
- To evaluate the effectiveness of ligand-based expression screening.
Main Methods:
- Screening of lambda gt11 expression libraries using recognition site DNAs.
- Adaptation of antibody probe-based screening strategies for DNA-binding proteins.
- Utilizing various probes, including binding site DNAs, proteins, and nucleotides.
Main Results:
- Successfully cloned numerous genes encoding transcriptional regulatory proteins.
- Demonstrated the effectiveness of the screening strategy across different structural motifs (helix-turn-helix, zinc finger, leucine zipper).
- Validated the use of DNA, protein, and nucleotide probes for library screening.
Conclusions:
- Ligand-based expression screening is a versatile and effective method for isolating genes of interest.
- This strategy significantly advances the ability to identify and study DNA-binding and transcriptional regulatory proteins.
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
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...
02:57Southern 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...