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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:
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.
These
cycling protocols are all performed in automated thermocyclers and
can be accomplished in a short time period.
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.
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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.
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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.
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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.
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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.
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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.
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Immunohistochemistry
Immunohistochemistry is a technique for identifying cellular or tissue constituents (antigens)
by means of antigen-antibody interactions, the site of antibody binding being identified
either by direct labeling of the antibody, or by use of a secondary labeling method.
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Conventional Chromosomal Karyotyping for detection of cancers.
Chromosomal analysis is aimed at studying: wild type transition of genes due to mutations by the virtue of complete inversion, base pair addition - deletions - or gene translocations. Cytogenetic studies may assist clinicians to look for the cause of Constitutional, Induced, Acquired abnormalities in the patients.
Clinical Cytogenetics analyses are performed on stained metaphase chromosomes to produce G banding specific to each chromosome; this allows for the detection of subtle changes in Chromosome structure. Varieties of other staining techniques are available to identify specific abnormalities. Stained preparations when examined under microscope, typically fifteen to twenty metaphases are scanned and counted, with at least five metaphases being fully analyzed. During complete analysis, each chromosome is critically compared for band, with it's homologous one. It is necessary to examine these many cells in order to detect clinically significant pattern of mosaicism.
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Fluorescent In Situ hybridization
The FISH technique has been carried out to study certain micro deletions that are beyond the resolution of other cytogenetic studies. The marker chromosomes that can not be identified by it's banding pattern, can be studied for various aberrations by applying high resolution FISH. The technique has gained wide acceptance in clinical Cytogenetics and in cancer biology. OncQuest, with its state-of-the-art laboratory performs FISH to locate a number of mutations in a gene including single base deletion. Fluorescent in situ hybridization has set up guidelines to identify aberrations in cancer causing genes as well as tumor suppressor genes. Radio labeled probe binds with the complimentary region of marker gene that is subsequently detected under fluorescent microscope by incorporating radio labeled antibody conjugates. The complex formed is physically mapped under fluorescent microscope to locate micro deletions, identify extra material of unknown origin and to spot subtle or complex rearrangements.
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