There is at least one intron, because the radiolabelled cDNA did not hybridize to the 1. Other introns may reside in the 2. Two methods that can be used to labed cDNAs are primer extension and nick translation. Primer extension uses an oligonucleotide primer and Klenow fragment to synthesize a labeled DNA strand. The "sticky ends" resulting from digesting with the enzymes are the same, but the actual restriction sites are different.
After isolating the plasmid, digest it with both enzymes, purify the 4. You can cut with both enzymes again after ligation — this would ensure that only a plasmid lacking both sites would transform the bacteria.
Walk to a clone that overlaps the end of a translocation or deletion this will show difference in Southern blots from organisms with and without the genetic abnormality. Use a clone from wild-type to pick up a junction fragment in the mutant strain. Use this fragment to pick up a clone at the other end of the chromosomal abnormality from a wild library. Run the mRNA out on a gel and transfer it to a blot this is called Northern blotting. Using primer extension or nick translation, label isolated genomic DNA and use it as a probe to hybridize to the blot.
The probe should hybridize to its corresponding mRNA, and the location of the probe on the blot should tell you the size of the mRNA. The rarer the site it recognizes, the smaller the number of pieces produced by a given restriction endonuclease.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. The cut is made between the adjacent G and C. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined.
However, many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single-stranded DNA. These are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. 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 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. 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. This virus is 48, base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs.
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. Gels may be discarded in regular trash receptacle. A description of how to use a micropipet can be found in Activity 2 - Gel Electrophoresis of Dyes. Although methylene blue dye is not as sensitive as ethidium bromide it may be used to stain the higher quantities of DNA that are used in this experiment.
Methylene blue is non-toxic but will stain clothes, hands, and equipment, so always wear gloves. Use the stain close to a sink and clean up spills immediately. Use distilled or deionized water to de-stain gels. Only use deionized water for making the 0. A single container of methylene blue dye should be all that is needed since it may be reused several times and disposed of down the sink.
Restriction enzymes require special care for handling and use. They lose activity unless kept frozen; exposure to warm temperatures for even a short time will result in loss of activity.
Using good sterile technique, aliquot samples for students, being careful to keep everything on ice until ready to be used. Enzymes should be stored in a foam container in the freezer non frost-free if available , along with the special buffer for each enzyme. The special buffers contain the salt and pH requirements for optimal activity of each enzyme. Since viruses have a relatively simple genome, scientists have studied their DNA and used this information to test theories and develop concepts that apply to the genetics of living organisms.
The information from the relatively simple virus genomes has been used to test theories and develop concepts that apply to the genetics of living organisms. Each enzyme recognizes a unique sequence of bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. This procedure is one of the most important in modern biology.
0コメント