Technology
A number of techniques are available to evaluate the structure of DNA, RNA or Proteins in cells. The most common methods used in the clinical setting include:
Polymerase Chain Reaction (PCR)
Real Time PCR
Probe-Hybridization Assays
Sequencing
Fragment analysis with fluorescent detection
Flow Cytometry
Immunohistochemistry
Conventional Chromosomal Karyotyping for detection of cancers.
Fluorescent In Situ hybridization
It is important to realize that a number of these different methodologies can be used to detect the same abnormality. The selection of one technique over another is often based on a variety of factors, such as sensitivity and specificity profiles, cost, turnaround time, and local experience.
Polymerase Chain Reaction (PCR)
The Polymerase chain reaction (PCR) is a technique that results in exponential amplification of a selected gene segment within minutes to hours. The reaction requires the presence of:
- Isolated genomic DNA or complementary DNA (RNA that has undergone in vitro reverse transcription to DNA)
- Essential components of DNA synthesis, including deoxynucleoside triphosphates (dNTPs), specifically designed oligonucleotide primers that flank the DNA segment of interest, thermostable DNA Polymerase, an appropriate buffer that contains magnesium chloride, and specific temperature cycling parameters that permit and control the amplification process.
After cycling is completed, the amplification products can be examined in various ways. Typically, the contents of the reaction vessel are subjected to gel electrophoresis. This allows visualization of the amplified gene segments (e.g., PCR products, bands) and a determination of their specificity. Additional product analysis by probe hybridization or direct DNA sequencing is often performed to further verify the authenticity of the amplicon.
Real Time PCR
The PCR reaction generates copies of a DNA template in an exponential manner. Due to presence of inhibitors, reagent limitation or accumulation of pyrophosphate molecules the reaction does not generate templates at an exponential rate throughout (the "plateau phase"). This is the most important reason that end-point quantitation of PCR products are unreliable.Real Time quantitative PCR allows the reliable detection and measurement of products generated during each cycle of the PCR process which is directly proportional to the amount of template prior to the start of the reaction. With the ability to measure the PCR products as they are accumulating, or in "real time", it is possible to measure the amount of PCR product at a point in which the reaction is still in the exponential phase and provides the most accurate and specific quantitation of the template.
Probe-Hybridization Assays
The common feature of probe hybridization assays is the use of a labeled nucleic acid probe to examine a specimen for a specific, homologous DNA or RNA sequence. The clinical probes are most often labeled with non-radioisotopic molecules such as digoxigenin, alkaline phosphatase, biotin, or a fluorescent compound. The detection systems are conjugate- dependent and include chemiluminescent, fluorescent, and colorimetric methodologies.Sequencing
Nucleic acid sequencing detects any mutation or variation within the region of interest. Single-base resolution capability makes sequencing the gold standard for mutation detection. Sequencing is performed using PCR to amplify the target nucleic acid sequence. Following PCR amplification, a dye-terminator sequencing reaction is performed for each sample.Fragment Analysis with Fluorescent Detection
For fragment analysis, PCR products are tagged with fluorophores and resolved by gel electrophoresis using a high resolution sequencing gel. An automated sequencer detects the alleles fluorescently. Reactions can be multiplexed, differentiating products by size and up to three fluorophores. Because multiple alleles and/or loci can be analyzed in a single reaction, fragment analysis is useful for DNA identity markers and simultaneous detection of multiple mutations.Flow Cytometry
Flow Cytometry is a technique to quantify the fluorescence and light scatter of particles in suspension. In the clinical laboratory, the particles are usually cells that fluoresce after binding to specific dyes, often fluorescently labeled antibodies. Flow cytometry is commonly used clinically to measure cell surface antigens and the DNA content of cells. The OncQuest Flow Cytometry Laboratory offers a full range of assays for immunologic monitoring, tumor phenotyping, and DNA content.