PCR

Examples of PCR in the following topics:

• Coupling Specific Genes to Specific Organisms Using PCR

  • PCR allows for the amplification and mutation of DNA and allowing researchers to study very small samples.
  • Without PCR, the studies we perform would be limited by the amount of DNA we were able to isolate from samples.
  • Common PCR protocols in labs today include knockout genotyping, fluorescence genotyping and mutant genotyping.
  • Researchers can use PCR as a method of searching for genes by using primers that flank the target sequence of the gene along with all other necessary components for PCR.
  • With the use of PCR, as few as 10 bacilli per million human cells can be readily detected.

• Multiplex and Real-Time PCR

  • The resulting cDNA serves as the template for the PCR reaction.
  • Real-time polymerase chain (RT-PCR) reaction, also called quantitative real-time PCR (qRt-PCR) is used to amplify and quantify targeted DNA molecules.
  • The procedure for RT-PCR follows the general principles of PCR, but the defining feature is the ability to detect amplified DNA as the reaction progresses in real time.
  • Real-time PCR can used to amplify low-abundance DNA templates.
  • Multiplex PCR is a challenging application that typically requires more optimization than standard, single amplicon PCR assays.

• DNA Sequencing of Insertion Sites

  • These include Southern hybridization, inverse Polymerase Chain Reaction (iPCR), and most recently, vectorette PCR to identify and map the genomic positions of the insertion sequences.
  • Inverse PCR, a commonly-used PCR method for recovering unknown flanking sequences of a known target sequence, uses a library of circularized chromosomal DNA fragments as a template and two outward primers located in each end of the known fragment for amplification .
  • Also, the length of each restriction DNA fragment containing a target sequence must be determined by Southern hybridization followed by sub-genomic fractioning before intramolecular ligation and PCR amplification.
  • These difficulties render Southern hybridization and iPCR impractical as techniques for quickly surveying repetitive elements in genomes.
  • Vectorette PCR (vPCR) is another method used to amplify unknown sequences flanking a characterized DNA fragment.

• Amplifying DNA: The Polymerase Chain Reaction

  • The polymerase chain reaction (PCR) is a method by which DNA is amplified.
  • PCR can be extensively modified to perform a wide array of genetic manipulations.
  • PCR is used to amplify a specific region of a DNA strand (the DNA target).
  • A basic PCR set up requires the following components and reagents:
  • This illustrates a PCR reaction to demonstrate how amplification leads to the exponential growth of a short product flanked by the primers. 1.

• DNA Analysis Using Genetic Probes and PCR

  • Example of genetic analysis method using PCR and immobilized oligonucleotide probes: The reverse dot-blot method has several unique properties that are valuable in a diagnostic setting: (1) The typing that results from a single sample can be located on a single strip.
  • This minimizes user labor as well as error potential and allows the use of standardized reagents. (3) Unlike dot-blot/oligonucleotide typing, only the PCR product is labeled, eliminating the potential problem of probes labeled to different specific activities.
  • The use of molecular technology in the diagnoses of infectious diseases has been further enhanced by the introduction of gene amplication techniques, such as the polymerase chain reaction (PCR) in which DNA polymerase is able to copy a strand of DNA by elongating complementary strands of DNA that have been initiated from a pair of closely spaced oligonucleotide primers.
  • In many PCR-based typing assays, the target DNA of interest is amplified and labeled by PCR, and the labeled products are hybridized to an array of immobilized diagnostic probes.

• Diagnosing Microbial Diseases

  • The identification of infectious agents is now often done by using molecular based techniques such as polymerase chase reactions (PCR).
  • PCR allows for the identification and testing for nucleic acids which are specific to the infectious agent.
  • The need of an infectious agent to amplify its own nucleic acids to ensure successful infection has allowed us to use PCR to detect the presence of these nucleic acids.
  • In combination with the advances made in genome sequencing, the tools and information needed to establish PCR as the gold-standard for diagnosing microbial disease is present.

• Obtaining DNA

  • Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (by a process known as Reverse-Transcription or RT-PCR) sequences prior to molecular cloning using primers or short DNA sequences specific for the region of interest.
  • This illustrates a PCR reaction to demonstrate how amplification leads to the exponential growth of a short product flanked by the primers. 1.

• Recombinant DNA Technology

  • Recombinant DNA technology also referred to as molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of a specific DNA sequence.
  • The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.
  • Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR) sequences prior to molecular cloning.

• Synthesizing DNA

  • This occurs in two fashions, by polymerase chain reaction (PCR) which is enzymatic and chemical synthesis.
  • PCR is covered in another atom.

• Specimen Collection

  • There are several types of specimens recommended for diagnosis of immunological diseases including: serum samples, virology swab samples, biopsy and necropsy tissue, cerebrospinal fluid, whole blood for PCR, and urine samples.
  • Refrigerated CSF is acceptable for a limited number of serologic tests; however, if PCR is to be performed for the viral panels, the specimen must be frozen and shipped on dry ice.