Unit 2 Chapter 9 Objectives

What is a Southern blot and how is it performed? What is a Northern blot? What is a Western blot?

A Southern blot is a process of detecting the presence of a specific nucleotide sequence in restriction fragments separated by electrophoresis through the use of DNA probes. The technique is named for Ed Southern, who developed it in 1975. After electrophoresis and staining, the DNA bands are transferred to a nitrocellulose filter by blotting. The nitrocellulose filter now contains DNA fragments positioned exactly as they are on the gel. The filter is exposed to a labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene, and the fragment containing the gene of interest is identified by a band on the filter. While a Southern blot is used to detect specific DNA molecules, a Northern blot is used to detect specific RNA molecules, and a Western blot is used to detect specific protein molecules (such as antibodies).

Define gene library.

A collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage.

Define protoplast fusion.

A method used in genetic engineering that consists of joining two cells by first removing their cell walls. When the protoplasts fuse, segments of the two chromosomes recombine and the recombinant cell grows a new cell wall.

Define clone.

A population of cells arising from a single parent cell.

What is a DNA probe? How does it work?

A probe is a labeled piece of DNA that is complementary to the sequence of the DNA of interest. The label allows you to visualize the probe. Probes are built with a DNA synthesis machine that can build short DNA molecules with any nucleotide sequence desired. When the labeled probe is mixed with DNA that has a complementary nucleotide sequence, and then the DNA is denatured so that the strands separate and the strands are allowed to reanneal, the probe can bind to (hybridize with) its complementary sequence. One way to make the label work is to use 32P labeled nucleotides (nucleotides with radioactive phosphorus in the sugar phosphate backbone). Through some manipulation, the DNA is placed on a piece of radiographic film and allowed to incubate long enough for the radioactive phosphorus to expose the film beneath the labeled DNA, and the film is then developed. Another option is to use a probe with fluorescent dyes attached. A light source is needed to excite the dye and a detector picks up the fluorescence emitted when the dye is excited.

Describe the use of restriction enzymes to generate recombinant DNA.

A restriction enzyme makes a staggered cut in a DNA molecule at the restriction site to create a fragment with two sticky ends. The same restriction enzyme is used to create a DNA fragment from another source. Because the same restriction enzyme was used, these two fragments have sticky ends with the same complementary base sequence. These two fragments can now join by base pairing and DNA ligase joins the backbones together, creating one whole molecule of recombinant DNA.

What is blue-white screening and how does it work?

Blue-white screening is a process that can be used to determine whether cells took up a blank vector or a recombinant vector. A vector containing the genes for ampicillin resistance (ampR) and the enzyme ß-galactosidase (lacZ - hydrolyzes lactose) is used. The restriction site is located at the lacZ gene, so any restriction fragment that manages to recombine with the vector will disrupt the lacZ gene and the plasmid can no longer code for ß-galactosidase. The plasmid vector and foreign DNA are cut with the same restriction enzyme and and introduced to bacteria. Cells are plated out on culture media that contains ampicillin and X-gal. Cells that weren't transformed will die from ampicillin poisoning, and cells that were transformed now have the ampicillin resistance gene and grow. Cells that took up non-recombinant (blank) vectors are ampicillin resistant, produce ß-galactosidase, and break down X-gal (which produces a blue color when broken down by ß-galactosidase), and the colonies turn blue. Cells with recombinant plasmids (with a restriction fragment inserted in the middle of the lacZ gene) can no longer make ß-galactosidase, cannot break down X-gal, and the colonies are white.

What is cDNA? How is it made? What is its significance?

Complementary DNA (cDNA) is made from mRNA by reverse transcription, using an enzyme (RNA-directed DNA polymerase - reverse transcriptase) that uses an RNA template to produce a complementary DNA molecule. Reverse transcription is essentially the reverse of the normal DNA-to-RNA transcription process. A DNA copy of mRNA is produced by reverse transcriptase. Following this, the mRNA is enzymatically digested away. DNA polymerase then synthesizes a complementary strand of DNA, creating a double-stranded piece of DNA containing the information from the mRNA. cDNA can be cloned in gene libraries, used for DNA sequencing, or used to genetically engineer bacteria to make eukaryotic gene products.

List all the ways you can think of that DNA can be inserted into cells.

Conjugation: transfer of information using sex pili Transduction: transfer of information by bacteriophages Transformation: uptake of naked DNA from outside the cell Protoplast Fusion: joining two cells by first removing their cell walls Microinjection: a micropipette punctures the plasma membrane of an animal cell, and DNA can be injected through it Use of a gene gun: shoots foreign DNA directly through the cellulose walls of plant cells

Define recombinant DNA.

DNA that contains genes from two or more different sources.

Define synthetic DNA.

DNA that is made artificially in the laboratory.

How can cells be screened to determine whether they took up a blank vector or a recombinant vector?

During DNA recombination, some plasmids won't recombine with restriction fragments and end up reannealing with themselves, forming "blank" vectors. Blue-white screening is a process that can be used to determine whether cells took up a blank vector or a recombinant vector.

Where does genomic DNA come from?

Genomic DNA is extracted from an organism that has the gene of interest.

Define genetic engineering.

Manufacturing and manipulating genetic material in vitro.

What are restriction enzymes? What is a restriction site? What is a restriction digest? What is a restriction fragment?

Restriction enzymes are enzymes produced by bacteria that function to protect bacteria from foreign DNA (like viruses). Restriction enzymes cut DNA at specific sites between nucleotides called restriction sites (the restriction enzyme recognizes the restriction site by its nucleotide sequence and then cuts the sugar phosphate backbone). A restriction digest is the result of using restriction enzymes to cut DNA into smaller manageable pieces called restriction fragments when recombining DNA from different sources.

What are sticky ends? What are they good for, and how are they used to generate recombinant DNA?

Sticky ends are overhangs on restriction fragments that occur when a restriction enzyme cuts somewhere other than the middle of its recognition sequence. These overhangs are "sticky" because they will hydrogen bond to another piece of DNA if it has the complementary base sequence. If you cut two different DNA molecules with the same restriction enzyme you'll get the same (complementary) sticky ends. The resulting restriction fragments can recombine, held together by hydrogen bonding at the complementary sticky ends. This is how you can get DNA strands from two different molecules to recombine and produce a hybrid or recombinant DNA molecule.

How can prokaryotic cells (like bacteria) express human genes when they have no mechanism for removing introns?

Synthesis of cDNA via reverse transcription

If you use DNA polymerase from a chimpanzee to make copies of a human gene and then insert the gene into a bacterial cell to make the gene product are the copies of the gene chimpanzee, human, or bacterial? Is the gene product chimpanzee, human, or bacterial?

The copies of the gene are bacterial because the introns have been removed. The product is human.

What is the polymerase chain reaction (PCR)? How does it work? If you use heat to denature the DNA template strands why doesn't the DNA polymerase denature?

The polymerase chain reaction (PCR) is used to make multiple copies of a desired piece of DNA enzymatically (in a tube). During PCR, the target DNA is denatured with heat (94-95° C for 1 minute) to separate the double stranded DNA template. It is then cooled to a temperature that will allow the short primers to anneal to the target DNA (but not cool enough to allow the template strands to reanneal - 55-65° C for 1 minute). Next, the temperature is increased to an optimal temperature for the polymerase to synthesize (72° C for 1 minute). Last, the entire cycle is repeated - each cycle produces 2 new copies from each template strand so the number of copies increases exponentially each time. The number of copies can be calculated, assuming faithful replication and an abundance of dNTPs and primers, as 2n copies where n = the number of cycles. For PCR, it is necessary to use heat stable DNA polymerase that will not denature when heated.

What is electrophoresis? What kinds of molecules can be separated by electrophoresis? How are they visualized?

The separation of substances, such as DNA fragments or proteins, by their rate of movement through an electrical field. This separation allows individual DNA fragments containing a gene of interest to be identified and isolated. Agarose, a seaweed extract available in powdered form, is mixed with buffer, heated to dissolve the agar, poured in a mold, and allowed to cool. As the agarose cools, a gel forms and the interior space resembles a meshwork or spider web at the molecular level. DNA molecules move through the the gel at different speeds based on size (small DNA fragments move more quickly than larger fragments). The gel is poured with a well-forming comb in place and once the gel solidifies, the comb is removed, leaving wells into which the DNA fragments are loaded. Before loading, the DNA fragments are mixed with a loading buffer that contains a mixture of dyes, glycerol, and typically EDTA, a chemical that binds ions and inhibits the activity of enzymes. The gel is then placed in a box that has electrodes at either end. Buffer is added to cover the gel. When power is supplied to the gel box, current flows through the buffer. DNA, being negatively charged, will migrate through the electrical field toward the positively charged electrode. DNA fragments of the same length will end up at the same place in the gel when the power is turned off. The DNA isn't visible as it runs on the gel, so in order to visualize the DNA after the fragments are separated, the gel may be stained with ethidium bromide, which will combine with the DNA strands and fluoresce orange when excited with ultraviolet light. Methylene blue is also sometimes used but isn't as sensitive as ethidium bromide (although it is much less toxic).

Define gene guns.

The use of a gene gun is a method of introducing foreign DNA into plant cells by literally shooting it directly through the cellulose walls. Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls. Some of the cells express the introduced DNA as though it were their own.

What are vectors? What is their purpose? What kinds of things are used as vectors? How are vectors designed to allow positive selection? How are cells selected based on whether or not they took up a vector?

Vectors are DNA molecules that transport foreign DNA into a cell. Plasmids and viruses are used as vectors. Properties required of a good vector: 1. Self-replicating 2. Small enough to manipulate outside the cell without breaking 3. Protected from degradation by host cell enzymes 4. Contain markers that allow identification of host cells that have successfully taken the vector into the cell When building or selecting a plasmid vector, it must contain at least one antibiotic resistance gene in order to allow positive selection. After cells are transformed (when they have been induced to take up the plasmid vector), they can be grown on culture media containing the antibiotic that the anitbiotic-resistance gene protects against. Cells that have the vector are resistant to the antibiotic and grow while cells that didn't take up the vector aren't resistant and don't grow.