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Tytuł oryginału: Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements.
Autorzy: Masternak Michał M., Przybylski Grzegorz K., Smoczkiewicz Paweł, Płotek Włodzimierz, Kowalczyk Dariusz W., Nowak Jerzy S.
Źródło: J. Appl. Genet. 2002: 43 (2) s.235-244, il., tab., bibliogr. s. 242-244
Sygnatura GBL: 305,055

Hasła klasyfikacyjne GBL:
  • genetyka

    Typ dokumentu:
  • praca doświadczalna
  • tytuł obcojęzyczny

    Wskaźnik treści:
  • ludzie

    Streszczenie angielskie: Since the invention of the polymerase chain reaction (PCR) several quantitative PCR-based approaches have been described. Recently, the real-time PCR method became a standard in quantitative PCR, althrough high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe to novel competitive PCR techniques, which were used to determine the frequency of T-cell receptor delta gene (TCRD) rearrangements in peripheral blood leukocytes. In the referrence gene competitive PCR (rgc-PCR) the rearranged TCRD gene competes with the reference gene (RAG1) for common reagents (dNTPs and Taq polymerase). The intensity ratio of amplification products, TCRD/RAG1, corresponds to the portion of cells containing a rerrangement. A series of reactions was performed, in which RAG1 primers were added to the PCR after different numbers of cycles. On the basis of the number of cycles needs to obtain equal band intensity, the frequency of cells containing a rerrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearragements. Vë1-Jë1 and Vë2-Jë1, compete for the common Jë1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlinke in the method using the internal competitor, the results are not affected by the quantity of quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable ang give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangement.

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