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.
For each lab group
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 0.1X TBE buffer to make this stain since the high chlorine levels of most tap water will damage the DNA. 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.
See description in Gel Electrophoresis of Dyes - Activity 2Enzymes
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.
The λ DNA used in this laboratory can exist as either a linear or circular molecule, creating some confusion when interpreting restriction digest results. By heating the sample to 60°C for 3 minutes, immediately prior to electrophoresis, the hydrogen bonds holding the ends of the linear DNA together in a circle will be broken.
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. One of the most studied viruses is called bacteriophage lambda (λ). Bacteriophage λ is a virus that infects bacterial cells.
Bacteriophage λ is a virus that attacks bacterial cells and is one of the most studied viruses. 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. The DNA of Bacteriophage λ is approximately 48,514 base pairs or 48.514 kilobase pairs in length while the human genome is approximately 3 billion base pairs.
This experiment uses special “restriction” enzymes that act as chemical scissors to cut λ DNA into pieces. Each enzyme recognizes a unique sequence of 4-6 bases along the DNA strand and cuts the strand at these sites - the first step in a process called restriction mapping. These smaller, specific sections of an organism’s DNA can then be studied in detail and an outline of the whole genome can be constructed. This procedure is one of the most important in modern biology.
The small fragments of DNA are separated by gel electrophoresis. The movement of the fragments will always be towards the positive electrode because DNA is a negatively charged molecule. The fragments move through the gel at a rate that is determined by their size and shape, with the smallest moving the fastest.
DNA cannot be seen as it moves through the gel. A loading dye must be added to each of the samples before it is pipetted into the wells. The progress of the dye can be seen in the gel. It will initially appear as a blue band, eventually resolving into two bands of different colors.
The faster moving, purplish band is bromophenol blue dye that migrates at roughly the same rate as a 300 base pair fragment of DNA. The slower moving aqua band is xylene cyanol, nearly equivalent to a 9000 base pair fragment. The faster moving band must move at least 4-7 cm from the wells to achieve the best separation of DNA for analysis. Care should be taken not to let the bromophenol blue band run off the end of the gel.
Following staining to locate the DNA, the gel is observed and the fragments appear as a pattern of bands. In this experiment, we will compare our banding pattern with a predicted result shown in figure 1.
Information may be provided by your teacher that details the process of isolating and analyzing these bands to create a DNA fingerprint.
The methylene blue dye will stain skin, clothes, and equipment. Always wear gloves and safety glasses. Do all staining in a central area near the sink.
Restriction enzymes cut at specific sites along the DNA. These sites are determined by the sequence of bases which usually form palindromes. Palindromes are groups of letters that read the same in both the forward and backwards orientation. In the case of DNA the letters are found on both the forward and the reverse strands of the DNA. For example, the 5’ to 3’ strand may have the sequence GAATTC. The complimentary bases on the opposite strand will be CTTAAG, which is the same as reading the first strand backwards! Many enzymes recognize these types of sequences and will attach to the DNA at this site and then cut the strand between two of the bases. The restriction enzymes which we used in this laboratory are EcoRI, HindIII and BamHI and their sequences are as follows, with the cut site indicated by the arrow.
This figure shows the size of each of the fragments/bands produced when λ DNA is cut with each of these restriction enzymes. The sizes were determined by comparison to a molecular ladder which has bands of known sizes when it is separated by electrophoresis at the same time as the digested λ DNA.
Restriction sites of Lambda (λ) DNA - in base pairs (bp)
The sites at which each of the 3 different enzymes will cut lambda DNA are shown in the maps Enzymes A, B and C below.
Protocol adapted from http://ceprap.ucdavis.edu/Equipment/Protocols/restriction_enzyme_analysis-methylene_blue_stain_03.pdfOther activities:http://www.biology.arizona.edu/human_bio/problem_sets/DNA_forensics_1/02Q.htmlUse problems 2 and 3 from Problem set 1.
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