Chapter 9

forensic microbiology

developed because hopsitals and food manufacturers can be sued in courts of law because microorganisms can be used as weapons; involves the identification and subtyping of infectious agents

clone

population of cells arising from one cell; each carries the new gene

bioinformatics

the science of understanding the function of genes through computer-assisted analysis. DNA sequences are stored in web-based databases referred to as GenBank

What is the purpose of a genomic library?

allows scientists to search for particular genes of interest.

gel electrophoresis

(1) DNA is digested by different restriction enzymes yielding thousand of fragments of various sizes (2) DNA Fragment (which have an overall neg charge) are put in a well at one end of the gel . An electric current is applied to the gel causing the different-sized pieces of DNA migrate through the gel at different rates (smaller fragments move faster than larger) (3) a blue dye (overall neg charge) is used as well and migrates toward the opposite charge (4) separated fragments are transferred onto a filter by blotting allowing identification of the different DNA fragments along the gel

Understand and be able to explain the basic steps involved in inserting a gene of interest into a plamid then into a bacterial cell.

(1) Restriction enzyme cuts DNA at recognition sites to obtain gene of interest (2) The same restriction enzyme is used on the selected vector to cut the DNA at the same recognition sites (3) These two DNA fragments join together at the "sticky ends" by base pairing (4) DNA ligase unites the backbone producing a molecule of rDNA (5) the rDNA plasmid can be inserted into a bacterial cell by transformation (bacteria cell takes up bacteria from surrounding environment after being treated with calcium chloride making them permeable to DNA molecules)

DNA fingerprinting

Analysis of DNA by electrphoresis of restriction enzyme fragments of the DNA; can be used to compare DNA from a crime scene to that of a suspect

Explain the difference between biotechnology & rDNA.

Biotechnology is the use of living organisms to modify human health and the human environment. Recombinant DNA technology is the process of isolating a gene and placing it within a vector--a typical way to use biotechnology.

Compare & contrast biotechnology, genetic modification, & recombinant DNA technology

Biotechnology uses microorganisms to make a product but unlike rDNA technology, the product is one that the organism produces naturally. Recombinant DNA technology involves transferring genes from one organism to another, or changing genes in an organism by altering their DNA to produce a desired product. Genetic modification involves altering an organisms characteristics (DNA) by the use of genetic engineering.

Genomic Library

Complete collection of cloned DNA fragments from an organism carried in plasmids or phages; extract an organisms DNA then the DNA is digested by restriction enzymes and the fragments are recombined into plasmid or phage vectors. These vectors are introduced into bacterial cell. Goal being a collection of clones, one for every gene of the organism

Be able to explain at least 2 applications for DNA technologies.

DNA technology can be used to produce insulin. Gene for insulin is combined with bacteria E.coli in rDNA to produce large quantities of insulin.Subunit vaccines (EX: hepatitis B) consist only of the protein portion of a pathogen and are made by genetically modifying yeasts. Since the protein is harvested from genetically modified cells and purfied for use as a vaccine there is not chance of becoming infected.

Steps in PCR.

Denaturation - The template strands that are bound together cannot be replicated, so the first step of PCR is to separate them by heating up the sample, breaking the hydrogen bonds between them. Annealing (recombine nucleic acid strands) - The sample is cooled just enough to allow the primers to bind to the ends of each of the two template strands Extension - DNA Polymerase attaches to the primers and makes a copy of each template strand. After the first cycle, there are 4 DNA strands. The process repeats with the 4 DNA strands, which will go on to make 8 strands, then repeat itself again to make 16 strands. PCR doubles the amount of DNA in a sample after each cycle, making it possible to obtain millions of copies of a DNA strand overnight.

restriction enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides; they are a protection mecahnism in bacteria against bacteriophages

genetic testing

Examining a person's DNA (genetic code) for any abnormalities; Determines if someone has a genetic disorder, will develop a disorder, or is a carier for recessive traits, usually done on susceptible individuals

Explain how one can specifically control which section of the genome is to be amplified.

It is the Polymerase enzyme that drives a PCR. A polymerase will synthesize a complementary sequence of bases to any single strand of DNA providing it has a double stranded starting point. This is very useful because you can choose which gene you wish the polymerase to amplify in a mixed DNA sample by adding small pieces of DNA complimentary to your gene of interest. These small pieces of DNA are known as primers because they prime the DNA sample ready for the polymerase to bind and begin copying the gene of interest. During a PCR, changes in temperature are used to control the activity of the polymerase and the binding of primers. (Using restriction enzymes you can isolate your gene of interest. Using PCR, add small pieces of DNA complimenary to your gene of interest (known as primers). Polymerase binds the DNA fragments and copying (amplifying) begins.)

List the 4 properties of vectors.

Must be capable of self replication; need to be of a size that allows for manipulation during rDNA procedures (smaller vectors more easily manipulated); preservation mechanism (ie: circular form of DNA protects DNA of vector); carrys a marker gene (helps when retrieving cells)

What is PCR & how is it used?

Polymerase Chain Reaction is a technique to make multiple copies of a piece of DNA enzymatically. Used to clone DNA for recombination, amplify DNA to detectable levels, sequence DNA, diagnose genetic disease, detect pathogens

Describe the use of plasmid & viral vectors.

R factor plasmids primarily used; plasmid DNA can be cut with same restriction enzymes as the DNA to be cloned. Viral DNA can accept much larger pieces of foreign DNA than plasmids can.

Define restriction enzymes, & outline how they are used to make rDNA.

Restriction enzymes are DNA cutting enzymes that protect a bacterial cell from bacteriophage DNA. They recognize & cut only 1 particular sequence of DNA. Bacterial DNA protects itself by addng a methyl group to cytosine. Sticky ends (cuts are not directly opposite each other) are most useful in rDNA because they can be used to join 2 different pieces of DNA that were cut by the same restriction enzyme (DNA Ligase used to unite the backbone) to form a molecule of recombinant DNA.

genomics

Study of whole genomes, including genes and their functions

gene therapy

The insertion of working copies of a gene into the cells of a person with a genetic disorder in an attempt to correct the disorder

Understand & be able to explain how transformed cells are selected for using either blue white screening or amp resistance.

The plasmid vector used contains the gene ampR, which codes for antibiotic resistance. The vector also contains the gene lacZ. These two genes are known as marker genes. The recombinant plasmid is introduced into a bacterium. The bacteria is cultured on a medium that contains the antibiotic ampicillin & X-gal, a substrate for lacZ. Bacteria that didn't pick up the recombinant plasmid won't grow because they don't have gene for antibiotic resistance. The bacteria that picked up the recombinant plasmid with the new gene inserted in the middle of the lacZ gene will not hydrolyze lactose & produce white colonies. The bacteria that picked up the original plasmid containing the intake lacZ gene will still hydrolyze lactose and turn blue.

Identify the roles of a clone & a vector in making rDNA.

The vector is self replicating DNA. It is inserted with the gene so it can carry the desired gene to the new cell. The clone is simply many copies of genetically identical cells each carrying the gene of interest / A desired gene is inserted into a DNA vector such as a plasmid or viral genome. The vector will insert the DNA into a new cell, which is grown to form a clone. The idea is that large quantities of the gene or the gene product can be harvested from the clone.

Explain two advantages and two problems with genetically modified organisms.

Two advantages: Human Insulin - better tolerated than insulin extracted from animal; produced by rDNA E. coli Bt cotton and Bt corn - these plants have been modified to carry the toxin-producing gene that kills insects that eat plants Two disadvantages:

What is 1 advantage of using E. coli for genetic engineering?

advantage of E. coli: it is easily grown and its genomics are well known disadvantage of E. coli: it produces endotoxins as part of its cell wall so they need to be eliminated from its products. It does not usually secrete protein products so cells must be lysed (broken down) to get the product

Making gene products with Saccharomyces cerevisiae.

also known as baker's yeast; best understood eukaryotic genome; more successful in expressing foreign eukaryotic genes than bacteria; yeasts are also more likely to continuously secrete the product

Making gene products with mammalian cells.

can be genetically modified to produce various products; best suited for making protein products for medical use because the cells secrete their products and there is a low risk or toxins or allergens

Making gene products with plant cells

can be grown in culture, altered by recombinant DNA techniques, & then used to generate genetically modified plants. These plants are useful for many valuable products (plant alkaloids- codiene, melanin). Advantages: large scale, low cost production and low risk of product contamination by mammalian pathogens or cancer-causing genes.

shuttle vectors

can exist in several different species

proteomics

is the science of determining all of the proteins expressed in a cell

transformation

enables cells to take up DNA from the surrounding environment; many cell types do not naturally transform. Soaking cells in a solution of calcium chloride makes the membrane more permeable to DNA allowing for transformation

recombinant DNA technolgy (rDNA)

insertion or modification of genes to produce desired proteins; microorganisms used as "factories" to make a product that the organisms don't naturally make

RFLP

restriction fragment length polymorphisms; the different sized pieces of DNA used in gel electrophoresis (created after DNA is first digested by a restriction enzyme)

vector

self replicating DNA used to carry the desired gene to the new cell; plasmids & viruses used are used as vectors

Biotechnologyy

the use of microorganisms, cells, or cell components to make a product (ie: foods, antibiotics, vitamins, enzymes)