[Club 100] Nucleic Acid Typing Method in Molecular Diagnosis

in hive-195150 •  3 months ago  (edited)

Good afternoon Steemians after a general introduction to molecular diagnosis now I will write on nucleic acid-based typing method. A nucleic acid is a constituent of the DNA (Deoxyribonucleic acid) molecule, made up of a Pentose or hexose sugar, a phosphate group and a nitrogenous base link between them by hydrogen bonds. (Hydrogen bonds are strong forces that link elements or molecules together). As examples of nucleic Acids we have Adenine, Guanine, Thymine, and Cytosine.

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Source structure of DNA and it's constituent Nucleic acids

Nucleic acid-based typing

Originally, nucleic acids were detected mainly by gene cloning strategies and hybridization procedures, which are laborious and time-consuming, limiting scientific investigations. The use of nucleic acid detection for the diagnosis of genetic and infectious diseases in clinical laboratories was made possible after the advent of PCR, a technique based on the amplification of nucleic acids by means of thermostable polymerase enzymes and a thermal cycler.

In this process, DNA duplex arrays are melted at high temperatures, and two oligonucleotides complementary to the target sequence of the flanking gene are specifically hybridized at a strict temperature depending on the sequence and length of the primer. Each PCR cycle consists of 3 basic steps: denaturation, hybridization, and polymerization. At the end of the PCR procedure, millions of identical copies of the original specific DNA sequence are generated.

When these copies have identical electrical charge and molecular weight, they should migrate simultaneously, forming a single band when applied to an electrophoretic gel. If the oligonucleotide primers used during PCR runs are labeled with a fluorescent dye, the PCR product can then be analyzed in a capillary electrophoresis instrument, which tracks the fluorescence of identical PCR sequences as they migrate.

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Source An example of a Polymerize Chain Reaction (PCR) Machine
The computerized instrument then generates a graph depicting a fluorescence peak at the migration location of the PCR product.
Variations of the technique include the use of a set of oligonucleotides to identify different organisms or variants in a single reaction on a biological sample. In addition, to increase sensitivity and specificity, nested and semi-nested PCR can be performed using an initial PCR product as a template.

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