Part+2_Mariah+Pierce

Part 2: Studying Expression and Function of Gene

I. Gel Electrophoresis A. __**Describe it**__ (what does it look like?) 1. Has a gel made of polymer 2. Gel placed into small plastic support and immersed in an aqueous sol. In a tray with electrodes at each end

B. __**Analyze it**__ (How does it function, what is it made of, components and parts?) 1. Uses a gel made of polymer, such as a polysaccharide 2. Gel acts as molecular sieve to separate nucleic acids or proteins by a. size b. electrical charge and other properties 3. Negatively charged nucleic acids (on their phosphate groups) travel toward positive pole in electrical field 4. Polymer fibers separate molecules by length, slowing down longer molecules 5. In all, separates mixture of linear DNA molecules into bands, each consisting of DNA mol of same length

C. __**Apply it**__ (what can you do with it and how can it be used) 1. Restriction fragment analysis a. rapidly provides useful info about DNA sequences b. DNA fragments produced by restriction enzyme digestion (cutting) of a DNA molecule are sorted by gel electrophoresis c. DNA molecules are separated by gel electrophoresis in restriction fragment analysis of both cloned genes and genomic DNA d. can distinguish between the normal and sickle cell alleles of the B-globin gene 2. Useful for comparing two different DNA molecules a. ex) two alleles of a gene b. tell if someone is heterozygous for carrier of mutant allele, but do not have purified B-globin alleles  i. Compares the alleles of the patient and alleles from a person with the disease and one without  D. __**Synthesize it**__; what does it make you think of?  1. Reminds me of forensics used to identify unknown DNA molecules  2. It is like a filter that arranges teh molecules by size and electrical charge  E. **__Argue__**:  1. Negative: restriction enzyme digested DNA stained with DNA-binding dye yields too many bands to distinguish individually  2. Positive: rapidly provides useful info about DNA sequencing – tell if samples came from same or different sources

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II. Southern Blotting- British chemist Edwin Southern A. __**Describe it**__ 1. Combines gel electrophoresis and nucleic acid hybridization 2. Includes alkaline sol., sponge, gel, nitrocellulose membrane, paper towels, and a heavy weight in that order

B. __**Analyze it**__ 1. Includes probe (same principal as hybridization) 2. Includes alkaline sol., sponge, gel, nitrocellulose membrane, paper towels, and a heavy weight in that order 3. Photographic film placed over blot to form an image 4. Function a. Each sample mixed with the same restriction enzyme b. restriction fragments separated by electrophoresis c. DNA transfer- capillary action pulls alkaline solution upward through gel, transferring DNA to a nitrocellulose membrane producing the blot; DNA denatures in process – Single strands of DNA stuck to nitrocellulose are positioned in bands corresponding to those on the gel d. Nitrocellulose blot exposed to sol. containing radioactively labeled probe e. Probe molecules attach by base pairing to restriction enzymes containing part of B-globin gene

f. Sheet of photographic film placed over= image corresponding to those bands containing DNA that base paired with the probe C. __**Apply it**__ 1. Allows us to detect those bands that include parts of the B-globin gene 2. Can differentiate a heterozygote (for sickle cell allele) from an individual homozygous for the normal allele 3. Can detect specific nucleotide sequence within a complex DNA sample 4. Useful for comparing restriction fragments produced from diff. samples of genomic DNA

D. __**Synthesize it**__ 1. It reminds me of a stamp because only the high points show up when being transfered to teh nitrocellulose

E. **__Argue__** 1. Can detect specific nucleotide sequences in order to identify unknown DNA sequences See more virtual lab []

III.DNA microarray assays A. __**Describe it**__ 1. DNA fragments placed on microarray where DNA fragments are fixed

B. __**Analyze it**__ 1. Consists of tiny amounts of a large number of single-stranded DNA fragments representing different genes fixed to glass slide in a tightly spaced array or grid 2. Nucleic Acid Hybridization lies at the basis of Microarray assays

C. __**Apply it**__ 1. Makes genome-wide expression possible 2. Can test thousands of genes simultaneously to determine which ones are expressed in a particular tissue 3. Can test them at diff developmental stages 4. Different environmental conditions 5. Various disease states 6. Contribute to better understanding of diseases

D. __**Synthesize it**__ 1. It is similar to a Computer chip holding all of this DNA information

E. __**Argue**__ 1. Better understanding of diseases = new diagnostic techniques Learn more []