Methods to study gene location, restriction mapping and other methods

Methods to study gene location, restriction mapping and other methods, Pulse field generator electrophoresis.

1. Methods to study gene location Sankalp UE211054

2. Restriction Mapping Used to determine the locations of specific restriction enzyme recognition sites along a DNA molecule, as well as the distances between these sites

3. Restriction Mapping Isolation of DNA: The first step in restriction mapping involves isolating the DNA molecule of interest which can be genomic DNA extracted from an organism, a plasmid DNA, or a specific DNA fragment obtained through techniques like PCR or cloning. Digestion with Restriction Enzymes: The isolated DNA is then treated with one or more restriction enzymes. These enzymes recognize specific DNA sequences, known as recognition sites or restriction sites, and cleave the DNA at or near these sites Separation of Fragments by Gel Electrophoresis: After digestion, the DNA fragments are separated based on their sizes using gel electrophoresis

4. Restriction Mapping Visualization of DNA Fragments: Once the electrophoresis is complete, the DNA fragments are visualized using a DNA stain, such as ethidium bromide or SYBR Green. These stains bind to the DNA molecules, allowing them to be visualized under UV light. Generation of Restriction Map: The pattern of DNA bands observed on the gel represents the sizes of the DNA fragments generated by restriction digestion. By analyzing the positions of these bands relative to known size markers, researchers can determine the sizes of the fragments. Mapping Analysis: Using the sizes of the DNA fragments obtained from the gel electrophoresis, researchers can construct a restriction map of the DNA molecule. This map shows the positions of the restriction sites along the DNA sequence and the distances between them

5. Pulse Field Gel Electrophoresis A variation of agarose gel electrophoresis Electric field driving the DNA molecules through the gel frequently changes position Can separate molecules as large as yeast chromosome (200-300 bp)

6. Breakdown of the mechanism Elongation: When the electrical field is first applied, the DNA molecules begin to elongate in the direction of the field Reorientation: The electric field then switches direction. Large DNA molecules take longer to reorient themselves to the new direction compared to smaller ones. Migration: Once reoriented, the DNA molecules can begin migrating again in the direction of the new field. Smaller molecules will be able to reorient and migrate faster than larger ones.

7. Breakdown of the mechanism Steps 2 and 3 are continuously repeated throughout the experiment. With each cycle, the larger molecules take progressively longer to reorient, resulting in their separation based on size