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The aim of the experiment is to identify which restriction enzyme, EcoRI or HindIII was used to cut the DNA plasmid.
A restriction enzyme is a protein that recognizes a specific nucleotide sequence and cuts the DNA only at that specific site. These restriction sites in the viral genome are cleaved by the bacterium’s restriction enzymes which fragments and destroys the DNA of invading bacteriophages before it can incorporate into the host’s genome and take over the cell.
A bacterium is immune to its own restriction enzymes due to the fact that bacterial restriction sites are highly methylated which makes them unrecognizable to the restriction enzyme. [1]
Scientists use restriction enzyme digest followed by electrophoresis as a way to separate DNA fragments.
After separating the DNA fragments through electrophoresis, it is transferred from the gel to a solid medium or membrane. This process is known as Southern blotting, named after Edwin Southern who developed this technique. [2]
To cut DNA at known locations, restriction enzymes that have been purified from various bacterial species are used.
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These enzymes are usually named after the bacterium from which they were first isolated. EcoRI cuts double-stranded DNA at the sequence GAATTC. The ends of a molecule cut by EcoRI have an overhanging region of single-stranded DNA which are known to be called sticky ends. [3] On the other hand, endonuclease HindIII is a type II restriction enzyme that recognizes and cleaves the palindromic sequence AAGCTT.[4]
DNA ligation occurs when DNA strands are covalently joined through the action of an enzyme, DNA ligase.
Sticky-ended molecules with complementary overhang sequences are said to have compatible ends which makes it possible for joining to form recombinant DNA. Two blunt-ended sequences are also considered to be compatible to join together, however they do not ligate together as efficiently. [3]
Bacteriophage lambda DNA is a double-stranded linear molecule. The digestion products are separated by agarose gel electrophoresis. [5] The negatively charged nucleic acid fragments migrate in an electric field within the agarose medium. The unknown molecular weight of each DNA fragment can be determined by comparing the electrophoretic mobility of the unknown fragment to that of a standard set of DNA fragments of known size. [6]
Materials and methods as per manual, however, 0.8% agarose gel was prepared in advance by the laboratory technician.
| Lane | Molecular weight markers |
|---|---|
| Lane 1 | EcoRI |
| Lane 8 | 1kb DNA ladder |
| Lane 15 | HindIII |
The sample used in this experiment can be seen at Lane 6.
As seen in the table of results above, the samples and markers can clearly be seen on the loaded agarose gel.
In comparison to the Lambda/ EcoRI standard ladder shown in fig.1, it can be distinguished that the sample loaded in Lane 6 is EcoRI. Fragments for the cut made by this restriction endonuclease are at 21226, 7421, 5804, 5643, 4878, and 3530 which were as expected. Based on the gel electrophoresis, it can also be identified that lane 6 performed similar fragmentation as lane 1 which was known to contain the lambda EcoRI restriction enzyme. In addition to that, it can clearly be seen that lanes 4 and 7 also contained EcoRI.
On the other hand, the lanes containing lambda HindIII restriction enzyme can also be clearly identified. Lane 15, which was known to contain this restriction enzyme, can easily be compared similarly to lane 3 and 10. HindIII restriction enzyme fragments were expected at 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125.
Overall, each lane can clearly be identified for the presence of either HindIII or EcoRI restriction enzyme; however, lane 14 shows unexpected results as it can be seen that the sample was smeared. A reason for this could be that the sample contained too much DNA. Another reason for this could be that the sample loaded contained a high salt concentration. As well as that, lane 9 and 11 also show undetectable bands showing no clear separations. A reason for this could be that there was not enough restriction enzyme in the sample, thus not cutting the DNA fragments of the sample. Lane 12 and 13 also show no fragmentation. A reason for this is that there was no DNA present in the sample loaded into the gel. [7] These errors are also caused by improper use of micropipettes which may have caused the DNA to degrade.
In general, the experiment was performed well yielding clear bands of fragmentation on the gel.
In conclusion, the experiment was a success as the restriction enzymes present in the lanes could clearly be identified according to the fragmentation cuts on the agarose gel. It can also be concluded that the sample used in this experiment contained the restriction enzyme lambda EcoRI.
The composition for the restriction enzyme HindIII is 10mM Tris-HCl (pH 7.3), 300mM NaCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 50% glycerol. [8]
EDTA is used as a chelating agent in gel electrophoresis. It chelates the Mg2+ ion required for enzyme DNAse as a cofactor. DNAse is an enzyme that cleaves DNA into small fragments. [9]
BSA is used during the digestion of DNA to prevent adhesion of the enzyme to reaction tubes, pipet tips, and other vessels. [10]
The unit of restriction enzyme is defined as the amount of enzyme required to produce a complete digest of 1 µg of substrate DNA in a total reaction volume of 50 µl in 60 minutes under optimal assay conditions. [11]
DNA molecules have a negative charge due to the phosphate groups in their sugar-phosphate backbone. [12]
EcoRI fragments are expected at 21226, 7421, 5804, 5643, 4878, and 3530. [14]
HindIII restriction enzyme fragments are expected at 23130, 9416, 6557, 4361, 2322, 2027, 564, and 125. [14]
The DNA ladder is a solution of different molecular weight fragments having double-stranded DNA used to determine the molecular weight or fragment size of the unknown PCR fragments. [13]
Lab Report: Restriction Enzyme Digest and Analysis. (2024, Jan 02). Retrieved from https://studymoose.com/document/lab-report-restriction-enzyme-digest-and-analysis
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