D nase, dms, microarray
- 1. PRESENTED BY: M.Sc II (SEM III) Microbiology DNase FOOTPRINTING,DNase FOOTPRINTING, DMS FOOTPRINTINGDMS FOOTPRINTING AND MICROARRAYAND MICROARRAY TECHNIQUETECHNIQUE
- 2. Contents 1.DNase footprinting a. introduction b. technique c. applications 2. DMS footprinting a. introduction b. technique c. applications 3. Microarrays a. introduction b. microarray technology c. applications
- 4. A DNase footprinting assay is a DNA footprinting technique from molecular biology that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short), to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern. This technique was developed by David Galas and Albert Schmitz at Geneva in 1977. INTRODUCTION
- 5. DNase I Footprinting assay is a method of studying DNA-protein interaction and identifying the DNA sequence to which a protein binds. First, a target DNA fragment about 100-300 bp in length is either PCR generated or cut from a vector and then uniquely labeled (at only one end) and incubated with protein, followed by controlled digestion with DNase I which cut the probe randomly but only once. TECHNIQUE The technique uses 32 P-radioactively labeled DNA..
- 6. The regions bound by proteins will be protected from DNase I digestion and will be shown as a blank area on the gel track. And the exact protein-bound sequence can be read out by comparing the location of the blank with the sequencing reaction. The digested DNA is recovered from the reaction and resolved on a polyacrylamide gel along with G+A chemical sequencing reaction which uses the same probe as template.
- 8. APPLICATIONS In vivo footprinting: In vivo footprinting is a technique used to analyze the protein-DNA interactions that are occurring in a cell at a given time point. DNase I can be used as a cleavage agent if the cellular membrane has been permeabilized. Quantitative footprinting: This footprinting technique can be modified to assess the binding strength of a protein to a region of DNA. Using varying concentrations of the protein for the footprinting experiment, the appearance of the footprint can be observed as the concentrations increase and the proteins binding affinity can then be estimated.
- 13. PAGE analysis of reverse transcription products: Sequencing lanes at the left are used to determine the position of modification for each experimental band. To the left and right of the gel is shown the sequence of the RNA, as determined from the sequencing reactions. The four lanes at the right show expected results from the experimental lanes. The ‘NO DMS’ lane is DMS-independent positions of reverse transcriptase stopping or pausing. In contrast, the bands that are enriched in the DMS treated samples (‘Unfolded’, ‘Folded’, and ‘+ protein’) reflect DMS modification.
- 14. DMS footprinting has been used to monitor a pH- dependent conformational change in the ribosome, RNA structural changes in response to changes in ionic conditions or mutation. It has been used extensively to monitor protein binding to ribosomal RNAs, as well as RNA-RNA associations, and binding of a small RNA regulator to an mRNA. It has also been used to monitor binding of small ligands to RNAs, notably the binding of antibiotics to ribosomal complexes. APPLICATIONS
- 16. An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. The large-scale genome sequencing effort and the ability to immobilize thousands of DNA fragments on coated glass slide or membrane, have led to the development of microarray technology. The concept of microarrays was first proposed in the late 1980s by Augenlicht and his colleagues. INTRODUCTION The core principle behind microarrays is hybridization between two DNA strands.
- 17. A microarray is a pattern of ssDNA probes which are immobilized on a surface called a chip or a slide. Microarrays use hybridization to detect a specific DNA or RNA in a sample. Fig- Robotic arm with spotting slides
- 18. Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases. DNA MICROARRAY TECHNOLOGY A DNA microarray is a collection of microscopic DNA spots on solid surface. Each spot contains picomoles of a specific DNA sequence, known as probes or reporters.
- 19. Fig- Array hybridization Each identified sequenced gene on the glass, silicon chips or nylon membrane corresponds to a fragment of genomic DNA, cDNAs, PCR products or chemically synthesized oligonucleotides and represents a single gene. Probe-target hybridization is usually detected and quantified by detection of fluorophore, or chemiluminescence labeled targets to determine relative abundance of nucleic acid sequences in the target.
- 20. Isolate a total RNA containing mRNA. Prepare cDNA from mRNA using a reverse-transcriptase enzyme. Each cDNA (Sample and Control) is labelled with fluorescent cyanine dyes (i.e. Cy3 and Cy5). Here, the labelled cDNA (Sample and Control) are mixed together. Purification. After purification, the mixed labelled cDNA is competitively hybridised against denatured PCR product or cDNA molecules spotted on a glass slide. TECHNIQUE Fig- Sample labeling
- 21. Slide is dried and scanned to determine how much labelled cDNA (probe) is bound to each target spot. Hybridized target produces emissions. Microarray software often uses green spots on the microarray to represent upregulated genes. Red to represent those genes that are downregulated and yellow to present in equal abundance. Fig- Gene chip showing different type of color spots
- 22. APPLICATIONS
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