JoVE Lab Manual
Lab Bio
Lab Bio
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DNA Isolation and Restriction Enzyme Analysis
DNA extraction is the removal and purification of DNA from cells. First, cells are lysed - broken open - usually though a combination of physical disruption and treatment with chemicals, such a detergent which dissolves the cell and nuclear membranes. SDS or sodium dodecyl sulfate is a commonly use detergent that works by solubilizing the proteins and lipids that make up these membranes. The contents can then float freely into the surrounding solution.
Next, DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is then added to the sample, which stabilizes the negatively charged phosphate groups in the backbone of the DNA and, after the addition of ice cold alcohol, precipitates the DNA out of solution. The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and resuspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications.
Some biotechnology applications, like DNA fingerprinting, which can identify novel patterns in DNA specific to an individual or individuals, involve the use of restriction enzymes. Restriction enzymes are molecules which interact with DNA and recognize specific sequences. Once their specific site is identified, they cut the DNA. This will result in the strand being cut into one or more linear pieces. If the DNA extracted was a plasmid, a circular piece of DNA most often found in bacteria, any cuts will result in the DNA forming a linear fragment or fragments. Different restriction enzymes recognize different DNA sequences, so using a combination of these can result in distinct fragments being produced.
In DNA fingerprinting, we can then examine these fragments using a technique called DNA or gel electrophoresis. To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is then added to the warm mixture and then this solution is poured into the casting mold. A comb is inserted to form the wells. When solidified, the gel is transferred to a gel box filled with buffer and the comb is removed. A reference mixture of dyed DNA fragments of known lengths, the DNA ladder, is added to one well and the dyed DNA samples of interest are loaded into the remaining wells. The box is connected to a power source and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel towards the anode, the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty.
When the run is complete the gel is exposed to ultra violet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands. Because DNA with different sequences will have cut sites at different locations, this can produce novel band patterns, or fingerprints, which can be used to distinguish individuals or variant DNA profiles.
In this lab you will perform DNA extractions using a buffer with and without SDS to assess the importance of detergents in DNA isolation and then digest plasmid DNA with different restriction enzymes to examine the resulting DNA profiles.
DNA Isolation and Restriction Enzyme Analysis
The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and forensics.
Isolating DNA from Cells
DNA isolation is a fairly simple procedure that requires few but essential steps: cell harvesting, lysis, protein degradation, and finally DNA precipitation. Generally, DNA can be harvested from small amounts of tissue, such as a single leaf of a plant, hair follicles of animals, or epithelial cells that can be easily sloughed off within the mouth with a simple saline solution. Since DNA is compartmentalized inside the nucleus or mitochondria within eukaryotic cells, scientists first break apart the cell and nuclear membrane to expose the DNA, which is done with the lysis step. However, once DNA is exposed, it can be degraded by heavy metal ions and extremes of pH. Therefore, a detergent-containing buffer solution is used to not only dissolve the cell membrane, but also to protect DNA from degrading elements in the solution. Moreover, DNA is an unusually long molecule that is condensed inside the nucleus by packing tightly around special proteins called histones. Therefore, an enzyme called Proteinase K, which degrades peptide bonds of proteins, is added to separate the DNA from histones and other proteins. Next, scientists separate DNA from the rest of the cellular extract by using its chemical properties. Due to the negatively charged structure of nucleic acids, Na+ ions in a sodium chloride solution bind to and integrate into entangled DNA strands. Once the DNA is clumped, it can be observed by the naked eye. DNA is insoluble in alcohol and readily precipitates at low temperatures, thus scientists add cold alcohol to precipitate DNA. Finally, the precipitated DNA can be dissolved and stored in deionized water or a mild buffer, free of any enzymes that can degrade nucleic acids, until it is used.
Quantifying DNA
Reactions that use DNA often require high purity and precise amounts of DNA. Therefore, following DNA isolation, scientists use a spectrophotometer to quantify the amount of DNA in their sample, as well as the purity of the DNA in the sample. A spectrophotometer measures the intensity of a light beam passed through a sample as a function of its wavelength. Nucleic acids absorb light at 260 nm, whereas proteins absorb light at 280 nm. By measuring the intensity of a light beam at 260 nm, scientists can determine the DNA concentration. Furthermore, by assessing the light intensity at both 260 nm and 280 nm, the purity of the DNA content can be determined. In a sample of pure DNA, the 260/280 nm ratio should approach the value of 2.
Restriction Enzymes
For a number of analyses, such as cloning, sequencing, and mapping DNA fragments and genomes, the isolated DNA needs to be cut at specific sequences using restriction enzymes. These enzymes are produced by bacteria as mechanisms of innate immunity against intruding viral DNA and function as molecular scissors. Hence, after restriction enzymes identify a viral DNA, they start to break it apart by cutting it into segments at certain nucleotide sequences they recognize. These nucleotide sequences are often six to twelve nucleotides long and are generally palindromic, which means that they have the same nucleotide sequence in the 3’-5’ and 5’-3’ directions. For instance, the restriction enzyme EcoRI cuts DNA at the sequence 3’...GAATTC…-5’, which reads the same in the 5’-3’ direction. Like EcoRI, there are hundreds of enzymes that correspond to many distinct palindromic sequences. Once DNA is cut by these enzymes, scientists can load the DNA onto a gel in an electrophoresis chamber and pass electrical current through the gel. DNA carries negative charges, which causes DNA fragments to migrate towards the anode. However, the pores of the gel slow down larger DNA fragments relative to smaller DNA sequences, thus the pieces separate according to their size. After the completion of the electrophoresis, DNA fragments can be visualized with the aid of dyes that specifically bind to DNA molecules.
Cutting DNA with restriction enzymes and separating DNA fragments with electrophoresis allows researchers to identify the sizes of unknown fragments relative to a standard ladder. These fragments of interest can then be further isolated and ligated into a plasmid vector for genetic cloning. Genetic engineering and laboratory analyses have been improved immensely with these methods. With DNA profiling, scientists are able to sequence genomes to further understand the workings of organisms and are also able to identify and characterize certain genes1.
DNA isolation and profiling are also practical for events directly impacting everyday life. For example, forensics scientists routinely analyze DNA samples to provide evidence for criminal and civil cases2. Similarly, healthcare workers extract and analyze DNA to check for paternity and genetic diseases. Recently, DNA ancestry analysis kits became popular by allowing users determine their genetic lineage, find relatives, and even discover their genetic disposition to various health conditions3. Finally, with advancements in biotechnology and genetic engineering, personalized medicine is gaining traction among clinical researchers to develop treatments by using an individual’s genetic data, paving the way to effectively treat patients without detrimental side effects4.
References
- Chambers, GK, et al. DNA fingerprinting in zoology: past, present, future. Investig Genet. 2014, Vol. 5, 3 doi: 10.1186/2041-2223-5-3.
- Roewer, L. DNA fingerprinting in forensics: past, present, future. Investig Genet. . 2013, Vol. 4, 22 doi: 10.1186/2041-2223-4-22.
- Kirkpatrick, BE and Rashkin, MD. Ancestry Testing and the Practice of Genetic Counseling. J Gen Counseling. 2016, Vol. 26, 1 (6-20).
- Hamburg, MA and Collins, FS. The Path to Personalized Medicine. N Engl J Med . 2010; , Vol. 363, 301-304.
