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Molecular Diagnostics
GenPath houses an advanced molecular laboratory to support each of our specialty medical diagnostics testing services. With a highly experienced staff of physician scientists and laboratory scientists, and working with our esteemed Scientific Advisory Board, GenPath can provide clinically relevant molecular testing services that are often not available at any other laboratory.
Technologies
Polymerase Chain Reaction (PCR)
PCR is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify (i.e., replicate) a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.
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Real-Time Quantitative PCR
Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique based on polymerase chain reaction, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.
The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
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Pyrosequencing
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle developed by Mostafa Ronaghi and Pål Nyrén (published in Analytical Biochemistry 1996 and Science 1998). For the history of the Pyrosequencing method see Methods Mol Biol. 2007. Pyrosequencing AB was started to commercialize the machine and reagent for sequencing of short stretches of DNA. Pyrosequencing AB was renamed to Biotage in 2003.
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Dideoxy DNA Sequencing
Dideoxy or chain-terminator or Sanger method of sequencing uses dideoxynucleotides triphosphates (ddNTPs) as DNA chain terminators. This method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing the four standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP). Incorporation of a dideoxynucleotide into the nascent (elongating) DNA strand terminates DNA strand extension, resulting in various DNA fragments of varying length. The dideoxynucleotides are added at lower concentration than the standard deoxynucleotides to allow strand elongation sufficient for sequence analysis.
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Gel Electrophoresis
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein molecules using an electric current applied to a gel matrix.[1] It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.
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SNP Array Genotyping
SNP arrays use hundreds to thousands of oligonucleotide probes attached to a solid surface (glass, silicon wafer, etc.) allowing for a large number of SNPs to be interrogated simultaneously (Rapley & Harbron 2004). To increase the accuracy of SNP detection, several redundant probes are directed to interrogate each SNP. Probes are designed to have the SNP site in several different locations as well as containing mismatches to the SNP allele. By comparing the differential amount of hybridization of the target DNA to each of these redundant probes, it is possible to determine specific homozygous and heterozygous alleles (Rapley & Harbron 2004).
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GenPath offers the following diagnostic testing services:
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