Nucleotide Synthesis and Purification - Report Sample

Introduction

The following are the materials and methods used in the six experiments in the purification and synthesis of a deoxyribonucleic analog labeled with a fluorescent dye, thus determining the ability to incorporate a probe into the nucleotides.

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Ion Exchange Chromatography

The ion-exchange chromatography separation procedure mainly comprises resins, which are critical for separation. The columns of the ion-exchange chromatography are packed in an ion exchanger. Ion exchangers are components of ion-exchange chromatography that attract each other at the ion-exchange chromatography stationary phase. The ion exchangers contain groups of ions that are linked covalently to the insoluble surface of the matrix. Therefore, the charged groups can be negatively or positively charged (Ion exchanger n.d). The resin used was DEAE-C Diethylaminomethyl cellulose, a good resin that is specifically used to purify nucleic acids. DEAE-C cellulose is a good resin since it generates high IC and has a high capacity of both nucleic acid and protein elution.

Labeling

The components used in this procedure were Base (A G C T U), deoxyribose sugar, and pyrophosphates in 5`-3` linkage. The manipulation process of nucleic acids included nucleases, ligases, polymerases, and restriction enzymes (blunt ends -for hanging ends, isoschizomers-cutting on same positions from 5'-3'directions and staggered -for cohesive ends). Ethidium bromide staining was used to label the nucleic acids. A tailing reaction procedure was used where a non-templated nucleotide was added to a blunt end of the nucleotide, spliced by the blunt restriction enzymes (Melissa Conrad Stöppler, 2018). The tailing procedure was used to A- tail the PCR nucleotide product produced through high fidelity.

Reverse Phase

The instrument conditions of the reverse chromatography should be modified at the beginning of the experiments to ensure that only a single gradient runs at a specific time so that the chromatography can achieve the highest selectivity possible. Thus, for the purity aspect, the instruments involved should be highly considered and modified appropriately. The reverse-phase chromatography methodology is where liquid chromatography in which molecules that are the nucleic acids are separated based on their hydrophobic interactions(Reversed-Phase Liquid Chromatography n.d). In this procedure, the ligands are attached to the stationary phase, and the solute molecules are attached to the stationary phase.

Fluorescence Spectroscopy

This is a sensitive biochemical tool used to analyze different spectral shapes and positions and study molecular structures. Fluorescent spectroscopy uses a dark background to maximize the view of the various elements in the background. The operation technique of fluorescence spectroscopy is such that it utilizes an abeam of light, which excites electrons of certain compounds and thus makes them emit light(Ranga. nr, 2019). The light emitted is directed towards a filter, which diverts to a detector. The detector quantifies the changes in a molecule, which are reflected in the graph.

Radioactivity

This is a quantification procedure that is used to quantify the number of radioactive precursors that are used in given procedures. Thus, the radioactive isotopes of high energy radiant are used to determine the reactions that synthesize new genetic material. The probes used are intercalated in the DNA sequence and there are fixed in between the phosphodiester bonds. A radioactive isotope probe used was 32P since it could easily intercalate in the phosphor diester bond. The liquid scintillation countermeasures a given sample's radioactivity by counting the resultant photon emissions from the sample (Ligasová,2018). A sample emits the radionuclides from light photons; beta particles are released and interact with the fluid. The light photons that are produced are measured. The scintillator vials used are used to provide high degree resistance to organic solvents.

Densitometry

This technique is used to determine the optical density in various light-sensitive materials(DNA Quantification by Gel Densitometry with Norgen DNA Ladders n.d). The range of light difference is recorded in a densitometer. DNA analysis can also be done through the densitometry technique. The DNA in densitometry is compared to that of flow cytometry.

Different labeling techniques were utilized in the above procedures and methodology of synthesizing and quantifying the nucleic acids. The methods or techniques of labeling can be limited to two dissecting groups: the radioactive and non-radioactive forms of labeling. The radioactive labeling utilized the32P- ATP -gamma (Ligasová,2018). The probe that comprises a phosphorous group is easily intercalated in the DNA nucleotides adjacent to the phosphodiester bond by utilizing an enzyme.

Conclusion

Finally, radioactive labeling is a technique that is easy and cost-effective; however, there is the danger of dealing with radioactive labels as they are hazardous. The other category utilized is the non-radioactive labels, also referred to as the chemical labeling procedure. This procedure uses labels commonly referred to as fluorescent tags that intercalate between the nucleotides and fluoresce to indicate a label. Other significant proteins are reactive to some groups, like streptavidin fluorophores that are also used as labels. The non-radioactive labels for nucleotides are used to determine the proteins that interact with nucleotides at a single molecule(Ligasová,2018). The assumption is that the non-radioactive labels are intercalated in every large nucleotide sequence. Moreover, for the short oligonucleotide sequence, the non-radioactive label is intercalated by the manufacturer.

References

DNA Quantification by Gel Densitometry with Norgen DNA Ladders. https://www.biocat.com/bc/pdf/DNA_Quantification_with_Norgen_DNA_Markers.pdf.

Ion exchanger.
https://www.britannica.com/science/ion-exchanger.

Jena Bioscience.
https://www.jenabioscience.com/

Ligasová, Koberna. DNA Replication: From Radioisotopes to Click Chemistry. Molecules. 2018 Nov 17;23(11). doi: 10.3390/molecules23113007.

Melissa Conrad Stöppler, M. D. (2018, June 12). Polymerase Chain Reaction (PCR): Steps in This DNA Test. https://www.medicinenet.com/pcr_polymerase_chain_reaction/article.htm.

New England Biolabs DNA and RNA Labeling.
https://www.neb.com/products/dna-modifying-enzymes-and-cloning-technologies/dna-labeling/dna-labeling

Ranga. nr. (2019, July 22). Fluorescence Spectroscopy Principle, Instrumentation, and Applications. https://www.studyread.com/fluorescence-spectroscopy/.

Reversed-Phase Liquid Chromatography.
https://www.sciencedirect.com/topics/nursing-and-health-professions/reversed-phase-liquid-chromatography.

Thermo Fisher Scientific Nucleic Acid Labeling Brochure.
https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-labeling.html.