Biotechnology http://library.thinkquest.org/28599/analogies.htm Biotechnology -- definition The use of living organisms, or substances from living organisms, to develop agricultural, medicinal, or environmental product or process. Some examples? Biotech tools Recombinant DNA: fragment of DNA composed of sequences originating from at least two different sources(organisms) http://www.cliffsnotes.com/study_guide/Recombinant-DNA-and-Biotechnology.topicArticleId-8524,articleId-8439.html Restriction endonuclease animation http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5 35::535::/sites/dl/free/0072437316/120078 /bio37.swf::Restriction Endonucleases Biotech tools Restriction endonucleases (restriction enzymes): enzymes that are able to cleave double stranded DNA into fragments at specific sequences Each type of restriction enzyme recognizes a characteristic sequence of nucleotides that is known as its recognition site How Restriction Enzymes Work Most recognition sites are four to eight base pairs long and are usually a complementary palindromic sequence The restriction enzyme EcoRI binds to the following base-pair sequence: 5´-GAATTC-3´ 3´-CTTAAG-5´ EcoRI scans a DNA molecule and only stops when it is able to bind to its recognition site. Once bound, it disrupts, via a hydrolysis reaction, the phosphodiester bond between the guanine and adenine nucleotides on each strand. Subsequently, the hydrogen bonds of complementary base pairs in between the cuts are disrupted The result is a cut within a DNA strand, producing two DNA fragments where once there was only one. Biotech tools EcoRI produces sticky ends Both fragments have DNA nucleotides that are now lacking their respective complementary bases SmaI produces blunt ends The ends of the DNA molecule fragments are fully base paired http://schoolworkhelper.net/2010/07/restriction-endonucleases-or-restriction-enzymes/ The following sequence of DNA was digested with the restriction endonuclease SmaI: 5´-TCGCCCGGGATATTACGGATTATGCATTATCCGCCCGGGATATTTTA-3´ 3´-AGCGGGCCCTATAATGCCTAATACGTAATAGGCGGGCCCTATAAAAT-5´ SmaI recognizes the sequence CCCGGG and cuts between the C and the G. (a) Identify the location of the cuts. (b) How many fragments will be produced if SmaI digests this sequence? (c) What type of ends does SmaI produce? Biotech tools: Methylases Restriction endonucleases must be able to distinguish between foreign DNA and the genetic material of their own cells. Methylases are specific enzymes that, in prokaryotes, modify the recognition site of a restriction endonuclease by placing a methyl group on one of the bases, https://netfiles.uiuc.edu/yxpan/www/Nutrigenegroup/Teaching.html Methylation Methylating a base prevents the restriction endonuclease from cutting the DNA into fragments. Methylases allow the molecular biologist to protect a gene fragment from being cleaved in an undesired location. Biotech tools: DNA Ligase Two fragments of nucleic acids generated using the same restriction enzyme, will naturally be attracted to each other at their complementary sticky ends Hydrogen bonds will form between the complementary base pairs, but this is not a stable arrangement. DNA ligase The phosphodiester linkage between the backbones of the double strands must be reformed. DNA ligase is the enzyme used for joining the cut strands of DNA together. http://www.biotechlearn.org.nz/themes/dna_lab/images/dna_ligation Biotech tools: gel electrophoresis Once a gene has been excised from its source DNA, it must be separated from the remaining unwanted fragments Gel electrophoresis takes advantage of the chemical and physical properties of DNA DNA properties DNA is negatively charged. The molar mass of each nucleotide pair is relatively consistent. The only difference between two fragments of DNA that are of differing lengths is the number of nucleotides. Gel electrophoresis DNA that has been subjected to restriction endonuclease digestion will be cleaved into fragments of different lengths. The shorter the fragment is, the faster it will travel because of its ability to navigate through the pores in the gel easily Larger fragments are hampered by their size. http://www.web-books.com/MoBio/Free/Ch9C.htm http://universe-review.ca/R11-16-DNAsequencing.htm Gel electrophoresis Gel electrophoresis takes advantage of DNA’s negative charge. A solution containing different-size fragments to be separated is mixed with a loading dye containing glycerol and is placed in a well in the gel The gel itself is usually a square or rectangular slab and consists of a buffer containing electrolytes and agarose, or possibly polyacrylamide. Using direct current, a negative charge is placed at one end of the gel where the wells are, and a positive charge is placed at the opposite end of the gel. Gel electrophoresis The negatively charged DNA will migrate toward the positively charged electrode The shorter fragments migrating faster than the longer fragments Small molecules found within the loading dye migrate ahead of all the DNA fragments. http://www.docstoc.com/docs/21969430/AGAROSE-GEL-ELECTROPHORESIS-TO-SEPARATE-DNA-FRAGMENTS-After Gel electrophoresis Once gel electrophoresis is complete, the DNA fragments are made visible by staining the gel The most commonly used stain is ethidium bromide which is a molecule that fluoresces under ultraviolet (UV) light and is able to insert itself among the rungs of the ladder of DNA. When the gel is subjected to UV light, the bands of DNA are visualized because the ethidium bromide is inserted among the nucleotides. http://en.wikipedia.org/wiki/Gel_electrophoresis Gel electrophoresis The size of the fragments is then determined using a molecular marker as a standard. The molecular marker, which contains fragments of known size, is run under the same conditions (in the same gel) as the digested DNA. The resulting graph can be used to determine the size of the Digital image of 3 plasmid restriction digests run on a 1% w/v unknown fragments through agarose gel, 3 volt/cm, stained with ethidium bromide. The DNA size interpolation. marker is a commercial 1 kbp ladder. Gel electrophoresis a researcher is able to estimate the size of a desired fragment the desired fragment can be excised out of the gel The region containing the desired fragment size can be purified for further use Genetic engineering experiment http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5 35::535::/sites/dl/free/0072437316/120078 /bio38.swf::Early Genetic Engineering Experiment Steps in cloning a gene http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5 35::535::/sites/dl/free/0072437316/120078 /micro10.swf::Steps in Cloning a Gene Biotech tools: plasmids Plasmids are small, circular, double-stranded DNA molecules that naturally exist in the cytoplasm of many strains of bacteria. Bacteria are able to express foreign genes inserted into plasmids http://lc-molecular.wikispaces.com/Isolating+Bli-1 Plasmids The bacterial cell benefits from the presence of plasmids. Plasmids often carry genes that confer: ◦ antibiotic resistance ◦ resistance to toxic heavy metals, such as mercury, lead, or cadmium Using a plasmid Restriction endonucleases are used to splice a foreign gene into a plasmid. Artificial plasmids have been engineered to contain a unique region that can be cut by many restriction enzymes. Recognition sites are present only once in the plasmid One cut results in the circular plasmid becoming linear. Biotech tools: transformation The introduction of DNA from another source is known as transformation A bacterium that has taken in a foreign plasmid is referred to as being transformed If a bacterium readily takes up foreign DNA, it is described as a competent cell. Bacteria can be chemically induced in the laboratory to become competent with the aid of calcium chloride. Transformation Bacterial cells are suspended in a solution of calcium chloride at 0°C. Positively charged calcium ions stabilize the negative charges of the phosphates on the membrane The low temperature “freezes” the cell membrane, making it more rigid. This stabilizes the cell membrane both physically and chemically, Next the plasmid DNA is introduced into the solution Transformation The entire solution is subjected to a quick heat shock treatment of 42°C that lasts for approximately 90 seconds The outside environment of the cell is now at a slightly higher temperature than the inside of the cell. The resulting draft sweeps the plasmids into the bacterial cell through pores in its membrane. Finally, the bacterial cells are incubated in a nutrient media suspension at a temperature of 37°C to recover Creating competent cells Electroporators—chambers that subject the bacteria to an electric shock which loosens the structure of the cell walls and allows foreign DNA to enter Modern electrical “gene guns” are used to “shoot” DNA through the cell wall and membrane of plant cells. Gene gun http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/McDonald/Gene_gun.html Electroporator http://biology200.gsu.edu/core/Manuals/Electroporator.html Biotech tools: selective plating Selective plating Selective plating is a method that can be used to isolate the cells with recombinant DNA If the transformation is a success, the bacteria will be able to grow on media that contain the antibiotic. If no growth is observed, the bacteria were not transformed and were eliminated by the antibiotic. Selective plating It is necessary to check that the foreign gene actually exists in the transformed bacteria. Colonies of bacteria are selected that have been transformed. Selective plating Individual colonies allowed to proliferate in liquid media until enough bacterial cells can be harvested to extract a suitable amount of plasmid DNA. The extracted plasmid DNA is subjected to a restriction enzyme digestion to release the cloned fragment from the vector. The DNA is run through electrophoresis to determine if the expected pattern of bands is observed on the gel The Major Steps in the Cloning of DNA 1. Generation of DNA fragments using restriction endonucleases 2. Construction of a recombinant DNA molecule • The target gene fragment is ligated to a DNA vector 3. Introduction into a host cell • Bacterial host cells can be manipulated to take up the recombinant DNA using electroporators, gene guns, or classical transformation protocols, such as calcium chloride The Major Steps in the Cloning of DNA 4. Selection • Cells that have been successfully transformed with the recombinant DNA must be isolated. • The desired cells are usually chemically selected by the presence of a marker (e.g. antibiotic resistance) on the vector. • Growth of colonies on media containing the chemical indicates successful transformation of the recombinant DNA vector. • Individual colonies are isolated from media containing the chemical and are grown in culture to produce multiple copies (clones) of the incorporated recombinant DNA.