Nucleotide Information and Courses from MediaLab, Inc.

Molecular diagnostic techniques utilize CE extensively. Automation, microvolume sample, increased sensitivity, immediate detection, and the computerization provided by CE enhance the analysis of nucleic acids. A multiple fluorescence detection system available with CE is also valuable.CE analysis of short tandem repeat polymorphisms is used in forensics, parentage testing, bone marrow engraftment analysis and other identification assays. Other testing for diagnosis of genetic diseases, oncology studies and DNA sequencing frequently utilize CE. DNA sequencing uses CE for separation of nucleotides labeled with multiple colored fluorescence dyes; CE and these markers enable computerized determination of the nucleotide sequence of DNA segments.

The basis of molecular testing lies in the genetic material of a cell. Both prokaryotic and eukaryotic cells possess nucleic acid. The two types of nucleic acid include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The foundational building blocks of both DNA and RNA include nucleotide bases of purines and pyrimidines. The unique sequencing of these nucleotide bases found in each strand of DNA or RNA contribute significantly to the language of cells. This cellular based language is responsible for many complex activities within the human body, including the synthesis of proteins.

DNA consists of the nucleotide bases: Adenine (A) Cytosine (C) Guanine (G) Thymine (T)RNA consists of the nucleotide bases: Adenine (A) Cytosine (C) Guanine (G) Uracil (U)

Term Definition Codon A three nucleotide base sequence that codes for an amino acid Genome The genetic code composed of 64 codons that code for 21 amino acids and 3 stop codons. (amino acids are the building blocks of proteins and stop codons stop the writing process much like a period at the end of a sentence) Nucleic acid Polymer made of monomers; two examples are RNA and DNA Transcription Process of transferring information from DNA into an RNA message Translation The formation of an amino acid from RNA Deoxyribonucleic Acid (DNA) A double-stranded polymer of nucleotides that houses genetic information Ribonucleic acid (RNA) Typically a single-stranded polymer that is much shorter than DNA but chemically similar with a few differences (e.g.- uracil replaces thymine). Replication Reproduction of DNA content from parent to daughter cell during cell division Amplification methods Techniques that increase the amount of the target, the detection signal, or the probe so that sequences are readily detected Fluorescence The emission of light at a longer wavelength when the light is excited at a shorter wavelength Oligonucleotide Short single-stranded nucleic acid Probe A nucleic acid used to identify a hybridization target Polymerase Chain Reaction (PCR) An amplification method performed in vitro

The reason to chose a particular molecular method can be influenced by disease detection, monitoring or therapy in certain patient populations. Molecular methodologies can be used to identify alterations or variations or changes in DNA sequencing that can cause disease. Sequence alterations that are known to cause disease are termed mutations. These changes or mutations can be applied to areas of the clinical lab such as infectious disease, paternity, genetic testing, and pharmacogenetics. Some of the more common alterations are:Deletion: a missing nucleotide or other portion of DNA sequence Insertion: an extra DNA nucleotide or other portion of DNA sequence Missense: a nucleotide or sequence substitution that codes for a different amino acidNonsense: a nucleotide substitution that ends in early termination of the protein manufacturing process; usually due to a stop codon.The most common alteration is a single base change or single nucleotide polymorphism (SNP)

Because hybridization involves nucleotide bases and the separation and joining or reannealing of strands, several environmental factors can influence this process: Temperature: If the temperature is too high, the strands melt. If it is too low, they might be forced together. The pH: A pH that is too alkaline will cause the strands to separate; too acidic and they are forced together. The guanine to cytosine ratio (G:C ratio): Since this bond is stronger than the other nucleotide bonds, if the G:C ratio in the desired target strand is high, the separation process may take longer.