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DNA Probing

DNA Probing. By: Jennifer Oxford, Jacob Carter, Elemuel Coleman. Table of Contents. What is a DNA Probe? DNA Probe Simplified Process of Searching for DNA/RNA Sequence Important Processes of Hybridization Southern and Northern Blotting Blotting Method Blotting Methods Recap

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DNA Probing

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  1. DNA Probing By: Jennifer Oxford, Jacob Carter, Elemuel Coleman

  2. Table of Contents • What is a DNA Probe? • DNA Probe Simplified • Process of Searching for DNA/RNA Sequence • Important Processes of Hybridization • Southern and Northern Blotting • Blotting Method • Blotting Methods Recap • Overview: Important Points

  3. DNA Probe • A molecule labeled with a radioactive isotope, dye, or enzyme that is used to locate a particular sequence or gene on a DNA molecule. • Also known as “hybridization probe” • DNA Probe is simply a sequence that is used to locate a specific DNA/ RNA fragment to take to PCR to be replicated.

  4. Simplified (Even More) • A DNA probe is a sequence of DNA bases that are complementary to a certain sequence that is present in the DNA strand. Wherever the DNA sequence is found, that is where the probe will show. For example, the sequence AGTTACT, the radioactive probe will show TCAATGA. From there, depending on the type of chemical the bands are viewed.

  5. Process of Searching for DNA/RNA sequence

  6. Process of Hybridization There are two vital steps of using hybridization: Denaturation and gel electrophoresis. Denaturation: Heating causes double helix strand to separate. Gel Electrophoresis-Used in Microbiology. Typically defined by the use of an electric field to move negatively charged nucleic acid molecules through an agarose. This is achieved by moving negatively charged nucleic acid molecules through an agarose gel. Shorter molecules move faster and migrate farther than longer ones.

  7. Southern Blotting and Northern Blotting Method Southern Blot-DNA Northern Blot-RNA Same method. However, the Northern blot is used specifically for RNA. • DNA Strands are cut into smaller fragments. • Gel Electrophoresis- Separates them further by size • Denaturation- to break DNA into single strands • Membrane placed on top and pressure is applied to get DNA from gel to membrane • DNA Membrane is exposed to DNA probe, which is examined using radiography that is tied to the probe for viewing.

  8. Blotting Methods

  9. Recap: DNA Probe and Processes • The DNA Probe is simply a specific sequence. There are methods of finding the specific sequence you are using. If you are searching for DNA- you southern Blot. If you are searching for RNA you use the northern blot. However, the DNA Probe can be either RNA or DNA

  10. DNA Probe: Scientific Case • In the Neonatal world, infections became noticeable in the Intensive Care Unit. The fungi was specified as Aspergillus Flavus. A DNA sequence from this fungi was probed. From there the sequence was detected in two different neonatal cases. The strain exists in a single laboratory and the sequence was located and destroyed.

  11. Overview: Important Points • DNA Probe= “Hybridization Probe” • DNA Probe is not a gel, it is a sequence used to locate a specific DNA Sequence • Southern Blotting-DNA; Northern Blotting-RNA • DNA probe is viewed using radiograph • Denaturation is the heating process that causes the DNA to break into single strand. • The cooling down and forming into regular shape is called annealing.

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