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Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) has become one of the most versatile and widely used techniques in Molecular Biology.  By combining specific primers and probes, genes or segments of nucleic acids (DNA/RNA) can be amplified for downstream applications in as little as an hour.  Because the PCR reaction results in an exponential replication of DNA, as little as a few copies of the gene of interest can be replicated into billions of copies by the end of the reaction.  These copies can be quantified against known standards analyzed concurrently to determine the starting amount of the gene of interest.

Since initial development of the method, the PCR process has been adapted and modified into numerous new methods including Real-time PCR (rt-PCR), Quantitative PCR (qPCR), Reverse transcriptase PCR (RT-PCR), Multi-plex PCR and Nested PCR, among others.  EAG utilizes rt-PCR for the determination of genetic sex in the Japanese Medaka and the African clawed frog as well as quantitative RT-PCR to detect levels of Vtg1 in the Japanese Medaka.

Ideal Uses
  • Gene expression and quantification
    • Vtg1 in Japanese Medaka (Oryzias latipes)
  • Genetic sex determination
    • DMY gene in Japanese Medaka ( latipes)
    • DM-W & DMRT genes in the African clawed frog (Xenopus laevis)
Technical Specifications
  • (2) 72-well Qiagen RotorGene Q thermocyclers (2-plex)
  • NanoDrop 2000
  • Thermal Mixer II for precise temperature control of DNA manipulations
Strengths
  • Minimal sample required for analysis
  • Specific amplification of genes of interest
  • Near real-time quantification of genes of interest
  • High through-put
  • Multi-plex/detection of numerous genes in the same assay
  • Real-time PCR eliminates the need of gel electrophoresis
Limitations
  • Primer/probe design dependent upon availability of sequenced gene/genome of interest
  • Gene section to be amplified in rt-PCR or qPCR should be between 80-200 nucleotides long.