Molecular Techniques Essay

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Ashley Switzer
Molecular Bio HW 2- Chart

Cation Exchange Chromatography-
Purpose/Function: Separates ions and polar molecules based on charged. Used in protein purification, water analysis, and quality control. Retains molecules on column based on ionic interactions. Cation exchange chromatography retains positively charged cations because the stationary phase is negative.
How Performed: A sample is introduced into the column. A mobile phase carries the sample into the column that contains the stationary phase (resin or gel matrix). The target cations are retained on the stationary phase. The retained analytes are then detected by conductivity or UV/Visible light.
Reference: http://www.sciencedirect.com/science/article/pii/S0021967397010303
What technique was used for: The researchers used cation exchange chromatography with different mediums to find three economically important proteins from bovine whey. They wanted to isolate IgG, lactoferrin, and lactoperoxidase based on their cation exchanges. The four medias were S-HyperD-F, S-Sepharose FF, Fractogel EMD SO3- 650 (s), and Macro-Prep High S Support. Each one of these mediums was used to find their binding capacity for IgG with a NaCl buffer.

Site-Directed Mutagenesis-
Purpose/Function: Used to make specific changes to the DNA sequence of a gene. Used for investigating structure and activity of DNA, RNA, and protein molecules, and for protein engineering.
How Performed: A short DNA primer is synthesized. The primer contains the mutation that is wanted and is complementary to the DNA around the mutation site. The primer is extended using DNA polymerase. The copied gene with the mutation is introduced to a host cell as a vector. The vector is cloned and the mutants are selected.
Reference: http://www.jbc.org/content/260/11/6518.short
What technique was used for: The researchers used site-directed mutagenesis to alter activity in proteins which are susceptible to chemical oxidation. From previous studies they noted that methionine 222 is a primary site of chemical oxidation (inactivation) of subtilisin. They prepared the other 19 amino acids for substitution at this site in the subtilisin gene. They expressed the mutant enzymes in Bacillus subtilis. They tested for the nonoxidizable and oxidizable amino acids by submitting them to a 1M H2O2 solution.

S1-Mapping
Purpose/Function: A method that uses mature mRNA to map particular DNA sequences using S1-nuclease. S1-nuclease removes single stranded DNA or ssRNA from DNA/RNA hybrids. Used to detect coding regions in DNA that have been hybridized. Leaves only coding DNA and can be used to find intron sites.
How Performed: A DNA probe is made that is labeled at only one end. The DNA probe is hybridized to the single stranded RNA. S1-nuclease is added to nature the remaining single stranded DNA or RNA. Length of protected part of the probe is determined through gel electrophoresis against marker DNA fragments of known length. Helps to determine the location of the transcription start site.
Reference: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC368769/
What technique was used for: They purified nascent transcripts of the simian virus 40. The isolated RNA molecules were isolated by sulfhydrylcellulose affinity chromatography. The RNA was hybridized to an end labled Hind III C probe fragments and subjected to S1-mapping to determine where the virus was spliced.

Nuclear run-on Analysis-
Purpose/Function- Used to identify genes that are being transcribed at a certain time. Allows changes in transcription rates to be measured. Uses radioactivity.
How Performed: Nuclei are isolated from appropriate cells using techniques that keep RNA polymerase bound to the genomic DNA. They are incubated with the four ribonucleotide triphosphates, with one that’s radiolabeled. Produces short radioactive RNA molecules. They are purified and the RNA of interest is determined by hybridization to appropriate DNA sequences
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