User:Heydi Dutan/sandbox

native gel electrophoresis: (checked) In the case of protein purification, Native gel Electrophoresis is a tool used to check for enzymatic activity and consequently the presence of the enzyme in the sample. For example, for the protein Alkaline Phosphatase, the staining solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-phospho-2-naphthoic acid-2’-4’-dimethyl aniline in Tris buffer. This stain is commercially sold as kit for staining gels. If the targeted protein is present, the reaction starts with the de-phosphorylation of 3-phospho-2-naphthoic acid-2’-4’-dimethyl aniline by alkaline phosphatase (water is needed for the reaction). The phosphate group is released and replaced by an alcohol group from water. The electrophile 4- chloro-2-2 methylbenzenediazonium (Fast Red TR Diazonium Salt) displaces the alcohol group forming the final product Red Azo Dye. As its name implies, this is the final visible-red product of the reaction. If the reaction occurs, our gel will have a band which will be visible at the same level of the commercially purified AP, proving the presence of the enzyme in our sample.

salting out (checked)

affinity chromatography (checked)

A current area of study and development are artificial ribonucleases, restrinction enzymes for RNA cleavage. Unfortunately the experiment hasn't been a complete success since the testing of these enzymes has shown slow rates. There is a ongoing parallel study in which scientist has developed PNA-based systems, also called PNAzymes. This PNAzyme has a Cu(II)-2,9-dimethylphenanthroline group that mimics ribonucleases for specific RNA sequence as it binds and cleaves a non based region of the targeted RNA. Its efficiency has been tested by exposing the sample to an excess of enzyme's substrate that have two potential cleavage sites, and as results, it was demonstrated the selectivity of cleavage to the respective design of the molecule. DNA- cleaving restriction enzymes have achieved a high level of development; now, PNAzymes could represent the premise for in vitro RNA restriction enzyme assemble.

(added to restriction enzymes/ with citation)

Added to PCR

Quantitative PCR: qPCR allows the quantification and detection of an specific DNA sequence in real time since it measures concentration while synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method are fluorescent dyes that are retained nonspecifically in between the double strands. The second method are probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place.An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique allows for mRNA to be converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR.

PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations.

libations of DNA cloning (g n g-) checked

Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centimeters and telomeres. Another characteristic that limits chances of success is large size of DNA sequence. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage λ can modified to successfully insert a sequence up to 40 kbp.

Added to topoisomerase (citation too) (checked )

Topologically linked circular molecules, aka catenanes, adopt a positive supercoiled form during the process of replication of circular plasmids. The unlinking of catenanes is performed by type IIA topoisomerase, which was recently found to be more efficient unlinking positive supercoiled DNA.The conformational properties of negative vs. positive supercoiled catenanes affects their features in respect to their corresponding enzymatic reaction catalyzed by topoisomerases. Experiments have demonstrated that positive supercoiled DNA provides a sharp DNA bend in the first bound DNA segment, which allows the topoisomerase to bind successfully and therefore carry on its enzymatic reaction to the following segment in an specific inside-to-outside matter. In the other hand, negative supercoiled DNA does not provide such bend and the access of the enzyme to the first segment is nearly impossible, therefore inhibiting unlinking.

DNA ligation. (added with citation) (checked)

The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. Mg(2+) is a cofactor for catalysis, therefore at high concentration of Mg(2+) the ligation efficiency is high. If the concentration of Mg(2+) is limited, the nick- sealing is the rate- limiting reaction of the process, and adenylylated DNA intermediate stays in the solution. Such adenylylation of the enzyme restrains the rebinding to the adenylylated DNA intermediate comparisonf an Achilles' heel of LIG1, and represents a risk if they are not fixed.