How many fragments will be generated in total upon digestion of a closed circular dna molecule and linear dna molecule with a same restriction enzyme having six recognition sites on both dna molecules?
Answers
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Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize. This recognition site or sequence is generally from 4 to 6 base pairs in length. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. The size of these fragments is measured in base pairs or kilobase (1000 bases) pairs.
Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested.
In the presence of specific DNA repair enzymes, DNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence. It doesn’t matter if the fragment that matches the cut end comes from the same organism or from a different one. This ability of DNA to repair itself has been utilized by scientists to introduce foreign DNA into an organism. This DNA may contain genes that allow the organism to exhibit a new function or process. This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area.
In this experiment, we will use restriction enzymes to cut up DNA from a small virus called Bacteriophage λ. This virus is 48,502 base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs. Since the whole sequence of λ is already known we can predict where each restriction enzyme will cut and thus the expected size of the fragments that will be produced. If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. This will result in fragments ranging in size from the smallest possible (all sites are cut) to in-between lengths (some of the sites are cut) to the longest (no sites are cut). This is termed a partial restriction digestion.
In this experiment, we will perform a full restriction digestion. After overnight digestion, the reaction is stopped by addition of a loading buffer. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed.
This laboratory will take approximately 3 days. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis. The gels may be stained overnight prior to photographing or recording results.
Objectives
Understand what a DNA restriction enzyme is and how it works.
Learn to use a micropipette.
Learn to separate DNA on an agarose gel using electrophoresis.
Understand how to use a restriction digestion map to identify a sample DNA.
Compare the λ DNA bands on a gel to the known λ DNA restriction map.
Materials
For each lab group
Four microtubes
Microtube rack
20-µl micropipette (or 10-µl micropipette) and sterile tips
Waterproof pen
Beaker or foam cup with crushed ice for the following
20 µl of 0.4 µg/µl λ DNA
2.5 µl BamHI restriction enzyme
2.5 µl EcoRI restriction enzyme
2.5 µl HindIII restriction enzyme
10 µl distilled water
Gloves
500-ml beaker (day 2)
Electrophoresis chamber (day 2)
Power supply (day 2)
20 µl 10X loading dye (day 2)
1.0% agarose gel (day 2)
Common Materials
Container with TBE solution (1X)
37°C water bath w/ floating rack
60°C water bath or saucepan on a hot plate (day 2)
Cooler with crushed ice
Freezer (non frost-free, if possible)
Camera if desired
Distilled water
0.002% methylene blue stain (day 3)
Advance Preparation
Day 1:
If you saved the 1X TBE solution from the Gel Electrophoresis with Dyes activity, reuse it for this laboratory.
Obtain enough crushed ice and ice containers (styrofoam cups) for each lab group.
Fill a pan with water and adjust it to 55°C on a hot plate
Fill a second pan with water and adjust it to 37°C on a hot plate while the students complete preparation of the restriction digests.
Reconstitute the lambda DNA with sterile distilled water to 0.4 µg/µl.
Aliquot lambda DNA, enzymes and loading dye for each group and keep in freezer until needed.