DNA ISOLATION AND PURIFICATION
Basic to all biotechnology research is the ability to manipulate DNA. First and foremost for recombinant DNA work, researchers need a method to isolate DNA from different organisms. Isolating DNA from bacteria is the easiest procedure because bacterial cells have little structure beyond the cell wall and cell membrane.
Bacteria such as E. coli are the preferred organisms for manipulating any type of gene because of the ease at which DNA can be isolated. E. coli maintain both genomic and plasmid DNA within the cell. Genomic DNA is much larger than plasmid DNA, allowing the two different forms to be separated. To release the DNA from a cell, the cell membrane must be destroyed.
manipulate 다루다
For bacteria, an enzyme called lysozyme digests the peptidoglycan, which is the main component of the cell wall. Next, a detergent such as sodium dodecyl sulfate (SDS) bursts the cell membranes by disrupting the lipid bilayer. For other organisms, disrupting the cell depends on their architecture.
detergent 세제
burst 터지다
disrupt 방해하다, 지장을 주다
lipid bilayer 지질 2중층
Tissue samples from animals and plants have to be ground up to release the intracellular components. Plant cells are mechanically sheared in a blender to break up the tough cell walls, and then the wall tissue is digested with enzymes that break the long polymers of lignin and cellulose into monomers.
mechanically 기계적으로
shear 깎다
DNA from the tail tip of a mouse is isolated after proteinase K degrades the tissue and detergent dissolves the cell membranes. Cells cultured in dishes are probably the easiest since they do not have cell walls or other structures outside their cell membrane. Detergent alone disrupts the cell membrane to release the intracellular components. Every organism or tissue needs slight variations in the procedure for releasing intracellular components including DNA. Once released, the intracellular components are separated from the insoluble remains such as the cellular membranes, bones, cartilage, and/or cell wall by either centrifugation or chemical extraction.
Detergent 세제
slight variation 약간의 변화
procedure 절차
Centrifugation separates components according to size, because heavier or larger molecules sediment at a faster rate than smaller molecules. In addition, materials that are insoluble in the liquid phase form aggregates that sediment to the bottom of a centrifuge tube faster.
sediment 침전물
aggregate 집합체
For example, after the cell wall has been digested, its fragments are smaller than the large DNA molecules. Centrifugation causes the DNA to form a pellet, but the soluble cell wall fragments stay in solution.
Another method of separating cellular components, chemical extraction uses the properties of phenol to remove unwanted proteins from the DNA. Phenol is an acid that dissolves 60% to 70% of all living matter, especially proteins. Phenol is not very soluble in water, and when it is mixed with an aqueous sample of DNA and protein, the two phases separate, much like oil and water.
properties 성질
aqueous 수분을 함유한
The protein dissolves in the phenol layer and the nucleic acids in the aqueous layer. The two phases are separated by centrifugation, and the aqueous DNA layer is removed from the phenol. Once the proteins are removed, the sample still contains RNA along with the DNA. Because RNA is also a nucleic acid, it is not soluble in phenol.
aqueous 수용액
Luckily, the enzyme ribonuclease (RNase) digests RNA into ribonucleotides. Ribonuclease treatment leaves a sample of DNA in a solution containing short pieces of RNA and ribonucleotides. When an equal volume of alcohol is added, the extremely large DNA falls out of the aqueous phase and is isolated by centrifugation. The smaller ribonucleotides stay soluble. The DNA is then ready for use in various experiments.
ELECTROPHORESIS SEPARATES DNA FRAGMENTS BY SIZE
Gel electrophoresis is used to separate DNA fragments by size (Fig. 3.1). The gel consists of agarose, a polysaccharide extracted from seaweed that behaves like gelatin. Agarose is a powder that dissolves in water only when heated. After the solution cools, the agarose hardens.
For visualizing DNA, agarose solidifies into a rectangular slab about 1/4 inch thick by casting the molten liquid into a special tray. Inserting a comb at one end of the tray before it hardens makes small wells or holes. After the gel solidifies, the comb is removed, leaving small wells at one end.
Gel electrophoresis uses electric current to separate DNA molecules by size. The agarose slab is immersed in a buffer-filled tank that has a positive electrode at one end and a negative electrode at the other. DNA samples are loaded into the wells, and when an electrical field is applied, the DNA migrates through the gel.
The phosphate backbone of DNA is negatively charged, so it moves away from the negative electrode and toward the positive electrode. Polymerized agarose acts as a sieve with small holes between the tangled chains of agarose. The DNA must migrate through these gaps. Agarose separates the DNA by size because larger pieces of DNA are slowed down more by the agarose. To visualize the DNA, the agarose gel is removed from the tank and immersed into a solution of ethidium bromide. This dye intercalates between the bases of DNA or RNA, although less dye binds to RNA because it is single-stranded. When the gel is exposed to ultraviolet light, it fluoresces bright orange. Since ethidium bromide is a mutagen and carcinogen, less dangerous DNA dyes such as SYBR Safe® are used in most laboratories now. This DNA dye is also excited by ultraviolet light, emitting a bright orange fluorescence. In Figure 3.1, the DNA fragments are visualized by a positively charged dye from the thiazin family. The dye interacts with the negatively charged backbone of the DNA and is a nontoxic alternative that does not require ultraviolet light sources. The size of DNA being examined affects what type of gel is used. DNA molecules of the same size usually form a tight band, and the size can be determined by comparing to a set of molecular weight standards run in a different well. Because the standards are of known size, the experimental DNA fragment can be compared directly. When DNA samples are separated by size through an agarose gel, DNA fragments from about 200 base pairs to 10,000 base pairs can be separated. For DNA fragments from 50 to 1000 base pairs, polyacrylamide gels are used instead. These gels are able to resolve DNA fragments that vary by only one base pair and are essential to sequencing DNA with the Sanger method (see Chapter 4). For very large DNA fragments (10 kilobases to 10 megabases), agarose is used, but the current is alternated at two different angles. Pulsed field gel electrophoresis (PFGE), as this is called, allows very large pieces of DNA to migrate further than if the current flows in only one direction. Each change in direction loosens large pieces of DNA that are stuck inside the gel matrix, letting them migrate further. Finally, gradient gel electrophoresis can be used to resolve fragments that are very close in size. A concentration gradient of acrylamide, buffer, or electrolyte can reduce compression (i.e., crowding of similar sized fragments) due to secondary structure and/or slow the smaller fragments at the lower end of the gel.
Fragments of DNA are separated by size using gel electrophoresis. A current causes the DNA fragments to move away from the negative electrode and toward the positive. As the DNA travels through agarose, the larger fragments get stuck in the gel pores more than the smaller DNA fragments. Pulsed field gel electrophoresis separates large pieces of DNA by alternating the electric current at right angles.