Please answer the data analysis questions #2, #5 and #6 at the end of the lab handout.I had to select a reference type in order to get a quote, but no references are needed.
Lab 7 BIOL1152: Basic Lab Techniques in Biology 1 Lab 7: DNA I Lab 7 Overview: Quiz (10mins) à Part 1: Restriction digestion (30 mins) à Part 2: Independent experiment (60mins) Practice loading during digestion Run gel for about 85 mins While running gel, work on final protocol independent experiments (60mins) Last 40mins view gel and analysis POSTLAB: gel HW & exp. plan Plasmids Plasmids are circular DNA that can replicate independently from the host’s chromosomal DNA. They are mainly found in bacteria, but also exist naturally in archaea and eukaryotes such as yeast and plants. In nature, plasmids provide one or more functional benefits to the host such as resistance to antibiotics, degradative functions, and/or virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number of the plasmid) and typically include a gene that is advantageous for survival, such as an antibiotic resistance gene. In contrast, plasmids utilized in the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally, lab-‐created plasmids have an origin of replication, selection marker, and cloning site (Figure 1). The ease of modifying plasmids and the ability of plasmids to self-‐replicate within a cell make them attractive tools for the life scientist and bioengineer. For instance, when scientists want to introduce a gene into a certain cell culture, they first introduce the gene into a plasmid and then they “grow” the plasmid in bacteria. What this means is that as bacteria reproduce, they make trillions of copies of the plasmid that can be collected by scientists. Basically, they use bacteria to get large quantities of the plasmid (Figure 2). The antibiotic resistant gene in the plasmid ensures that only bacteria containing the plasmid can survive on agar plates containing the antibiotic. Figure 1. Plasmid map depicting features with descriptions in the table. Lab 6 BIOL1152: Basic Lab Techniques in Biology 2 DNA splicing, the cutting and linking of DNA molecules, is one of the basic tools of modern biotechnology. The basic concept behind DNA splicing is to remove a DNA fragment of interest — let’s say a gene — from the chromosome of one organism and to combine it with the DNA of another organism in order to study how the gene works. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes have been given to people with nonfunctional genes, such as those who have a genetic disease like cystic fibrosis. Restriction Enzymes — Molecular Scissors Restriction enzymes, also known as restriction endonucleases, are proteins that cut DNA at specific sites and thus, they act as molecular scissors, making cuts at the specific sequence of base pairs that it recognizes. The sequence that they recognized is called a restriction site. The three-‐dimensional structure or shape of a restriction enzyme allows it to fit perfectly in the groove formed by the two strands of a DNA molecule. When attached to the DNA, the enzyme slides along the double helix until it recognizes a specific sequence of base pairs, which signals the enzyme to stop sliding. The enzyme then chemically separates, or cuts, the sugar-‐phosphate backbone of DNA. Viruses called bacteriophages are major enemies of bacteria. These viruses infect bacteria by injecting their own DNA into them, hijacking the bacterium’s replication machinery, to produce viral DNA. Bacteria have responded by evolving a natural defense: they produce restriction enzymes that cut up and destroy the invading DNA. Bacteria prevent digestion of their own DNA by modifying certain DNA bases within the specific enzyme recognition sequence, which allows them to protect their own DNA while cutting up foreign DNA. This could be considered a very primitive immune system. Restriction enzymes search the viral DNA for specific palindromic sequences of base pairs (Figure 3). Some restriction enzymes may leave a short length of unpaired nucleotide bases, called a “sticky” end, at the DNA site where they cut, Figure 2. Bacteria are used to propagate and create large quantities of plasmids. When bacteria divide they create a copy of the plasmids inside them. You can begin with a single bacterium that contains your plasmid. After a single day of culture, you can have millions of bacteria cells resulting from cell division each with a copy of your plasmid. Figure 3. A palindromic sequence of bases reads the same forwards as it does backwards on the opposite DNA strand (Example: 5’-‐GAATTC’3’). The actual palindromic sequence of DNA is called a restriction site, and DNA is cut at these sites. Lab 6 BIOL1152: Basic Lab Techniques in Biology 3 whereas other restriction enzymes make a cut across both strands creating double stranded DNA fragments with “blunt” ends. There are thousands of restriction enzymes, and each is named after the bacterium from which it is isolated. For example: EcoRI = The first restriction enzyme isolated from Escherichia coli bacteria HindIII = The third restriction enzyme isolated from Haemophilus influenzae bacteria PstI = The first restriction enzyme isolated from Providencia stuartii bacteria As you can see in the list below, each restriction enzyme recognizes one specific restriction site and cuts that sequence DNA always the same way leaving either 3’ overhangs, 5’ overhangs or blunt ends. If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme will make a cut at each of those sites, resulting in multiple fragments of DNA. Therefore, if a given piece of DNA is cut with a restriction enzyme whose specific recognition sequence is found at five different locations on the DNA molecule, the result will be six fragments of different lengths. The length of each fragment will depend upon the location of restriction sites on the DNA molecule. Electrophoretic Analysis of Restriction Fragments A DNA fragments that has have been cut with restriction enzymes can be separated by size using an agarose gel and a process known gel electrophoresis. The term electrophoresis means to carry with electricity. With the help of a loading solution (see ‘loading dye’, below), DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively charged, they will be drawn toward the positive pole (cathode) when placed in an electric Lab 6 BIOL1152: Basic Lab Techniques in Biology 4 field. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs. Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single bands of DNA. These bands will be seen in the gel after the DNA is stained (treated with a chemical that will allow DNA visualization). An analogous situation is one where all the desks and chairs in the classroom have been randomly pushed together. An individual student can wind his/her way through the maze quickly and with little difficulty, whereas a string of four students would require more time and have difficulty working their way through the maze. Making DNA Visible Staining the DNA pinpoints its location in the gel. To visualized DNA fragments, Ethidium Bromide is added to agarose gels. This compound intercalates between nucleotides and is lights up when illuminated with UV light. The solution you load into the gel before this though is colorless. A loading dye containing two blue dyes is added to the DNA solution. The loading dye does not stain the DNA itself but makes it easier to load your sample into the gels and monitor the progress of the DNA electrophoresis. The dye fronts migrate toward the positive end of the gel, just like the DNA fragments. The “faster” dye co-‐migrates with DNA fragments of approximately 500 bp, while the “slower” dye co-‐migrates with DNA fragments approximately 5 kb in size. Part 1: Restriction