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Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis.

An amphoteric molecule has the ability to be negatively or positively charged. Changing the pH using buffers will alter the charge and magnitude of the charge. A molecule with this amphoteric ability is sometimes referred to as an ampholyte or even by the older term, zwitterion.Proteins with their ionizable amino and carboxyl groups are amphoteric. Nucleic acids (deoxyribonucleic acid or DNA and ribonucleic acid or RNA) are charged and thus are amphoteric.

More separations are also achieved with layers of polyacrylamide gels each with a different pore size. The gels can be horizontal or vertical slabs or incorporated into vertical cylinders or rods. Varying the pore size in each layer is significant especially if very small pore sizes are created. DNA of 100 base pairs (bp) or less can be separated.Common applications of PAGE are separation of proteins and nucleic acids. Polyacrylamide gels are also used as the medium in several other types of electrophoresis described in this section.

Minute-size fractions achieved in two-dimensional electrophoresis, IEF and PAGE with SDS, and bands from electrophoresis of nucleic acids are detected differently than protein electrophoresis fractions. Labeled polypeptide probes are used to detect these proteins; labeled single-stranded nucleic acid fragments are used for the detection of nucleic acids. Each probe is made with a label designed to generate a detectable signal. The label is bound to a probe and a system is created such that the signal is visualized when the probe is bound to the target.The most common labels are radioactive isotopes and fluorescence dyes. Chemiluminescence and color or ultraviolet absorbance are also used.

Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting)does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest.

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.

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

Some methodologies include: Polymerase Chain Reaction (PCR) Ligase Chain Reaction (LCR) Transcription Mediated (TMA) Transcription Based (TAS) Strand Displacement (SDA) Branched DNA (bDNA) Loop mediated (LAMP) Nucleic acid sequence based (NASBA)

These methodologies use principles that amplify or multiply the target of interest, usually incorporating an enzyme to produce millions or billions of copies in a relatively short time.Some enzymes used in amplification include: DNA ligase DNA polymerase RNA polymerase Reverse transcriptase Alkaline phosphatase Cleavase Note: the steps in amplified testing will vary depending on the target amplified, requirement for thermal cycling and detection techniques.

Hybridization is the pairing or annealing of two strands of DNA. Hybridization is therefore based on the formation of double stranded hybrids from single stranded nucleic acids. These double stranded hybrids form under precise conditions and are detected using probes. A probe is a set of nucleic acids of known identity which seeks out the target of interest. Depending on the detection technique, probes and/or targets can either be labeled or unlabeled and the reaction can take place with one attached to a matrix or in solution, thus dividing the techniques into two broad categories: Solid phase Solution phase

Southern Blot: Employs a restriction endonuclease enzyme to extract DNA from the cells. DNA detection is done using agarose gel electrophoresis.Fluorescent In Situ Hybridization (FISH): Uses RNA Northern Blot or DNA Southern Blot techniques to detect targets of interest in cytology/histology specimens or other nucleic acid variations. DNA fingerprinting: Restriction Fragment Length Polymorphism (RFLP): Cuts long DNA into shorter fragments before detection to isolate changes or polymorphisms. These can either be detected by Southern Blot or by Polymerase Chain Reaction (PCR).

HIV consists of two basic components: a core of nucleic acid, called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The capsid is made up of subunits called capsomeres.

The HIV type-1 belongs to the Family Retroviridae and consists of two basic components: a core of ribonucleic acid (RNA), called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The capsid is made up of subunits called capsomeres.