Publish Your Research Online
Get Recognition - International Audience
Request for an Author Account | Login | Submit Article
|HOME||FAQ||TOP AUTHORS||FORUMS||PUBLISH ARTICLE|
DNase Foot Printing: Identification of Protein Binding Site of DNABY: Muniba Safdar | Category: Genetics | Submitted: 2010-07-27 06:21:15
Article Summary: "What is DNA foot printing? It is a technique used to identify protein binding site of DNA. As DNA exists in a cell in the form of nucleoprotein complex. Proteins interact with the DNA for the protection of DNA. It means there is DNA-protein interaction in the cell. There are specific sites where proteins bind..."
DNA foot printing is a technique used to identify protein binding site of DNA. As DNA exists in a cell in the form of nucleoprotein complex. Proteins interact with the DNA for the protection of DNA. It means there is DNA-protein interaction in the cell. There are specific sites where proteins bind. Now, we have to identify that site.
Step 1: DNA strand
• Our source is recombinant DNA/cloned DNA.
• Usually, we consider 100-300 base pairs.
• We will take different fragments starting with same point and ends with the same pint.
• After getting a strand of DNA, we will label only one strand.
• Labeling of DNA will be at 5´ end.
• Label with phosphorous 32 (P32) and phosphorous 35 (P35).
• P35 gives glow to DNA.
Step3: DNase I
• DNase I, an enzyme, which only cuts one strand from 5´ to 3´ end.
• Best cleavage agent.
• After labeling of DNA, they are then added with DNase I.
• It randomly cuts the strands.
• Concentration of DNase I should be low so that only one cut in one molecule.
Step4: Run on gel
• Now, cleaved DNA in the absence of DNA binding protein is compared to the cleaved DNA in the presence of a DNA binding protein.
• That site will be protected from enzymatic cleavage where protein bound to the DNA.
• Protection from enzymatic cleavage would result in a fine clear are on the gel which is referred to as "foot print of a protein".
• Through this technique we come to know which sequence is present here.
If we do some modifications in the above steps we will get a technique called EMSA, Electrophoretic Mobility Shift Assay. In this technique
• Particular length of DNA is used.
• Restriction fragments.
• Mild concentration of DNase I.
• Gel used is of good/high resolution that can differentiate small distances.
• We can use urea in a gel to denature.
• Only labeled fragment will glow under radiography.
• Protein has to give more time when bind with DNA.
Why we prefer this technique?
• If we do not have DNase I we can use chemicals, guanosine or any other reagent.
• If there is a site present between two base pairs it can determine that site.
Why we do not prefer this technique?
• It is very tedious (time consuming).
• Technically demanding. For example, pH changes, cut site, etc).
• We are basically using DNases and its cofactor is Mg++ and we must have to use it, because without this cofactor it will not work.
About Author / Additional Info:
Comments on this article: (0 comments so far)
• Surgery, Radiotherapy and Chemotherapy Followed by Tumor Immunotherapy?
• Laboratory Data For Blood Test
• Laboratory Data For Blood Test
• Compost Input: A Technology For Soil Restoration
Latest Articles in "Genetics" category:
• The Science and History of Genetics. How It Predicts the Genetic Code
• Telomeres: Is It Responsible For Ageing and Cancer?
• Human Genetic Engineering,its Methods and Ethics
• Gene Mutation And Cancer
• DNA Technology Used in Forensics
• DNA Fingerprinting: Uses and Methods Involved
• Treatment of Genetic Diseases by Gene Therapy
• Human Intelligence and Genetics
• Ethical Issues Related to Human and Animal Cloning
• Mitochondrial DNA and Forensic
• DNA Footprinting and Gene Sequencing
• Biotechnology and Types of Cloning
• Designer Babies:Method and Ethical Issues
• Prenatal Diagnosis: Non-invasive and Invasive Techniques
• What are the Benefits of Genetic Engineering?
• The Advantages and Disadvantages of Genetic Engineering in Humans
• Types of Genetic Disorders
• Bovine Somatotropin: A Growth Hormone
• Advantages and Disadvantages of Genetically Modified Food
Important Disclaimer: All articles on this website are for general information only and is not a professional or experts advice. We do not own any responsibility for correctness or authenticity of the information presented in this article, or any loss or injury resulting from it. We do not endorse these articles, we are neither affiliated with the authors of these articles nor responsible for their content. Please see our disclaimer section for complete terms.
Copyright © 2010 biotecharticles.com - Do not copy articles from this website.
ARTICLE CATEGORIES :
| Disclaimer/Privacy/TOS | Submission Guidelines | Contact Us