Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements.
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, although high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe two 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 reference 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 rearrangement. 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 needed to obtain equal band intensity, the frequency of cells containing a rearrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearrangements, Vdelta1-Jdelta1 and Vdelta2-Jdelta1, compete for the common Jdelta1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlike in the method using the internal competitor, the results are not affected by the quantity or quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable and give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangements.
Masternak
Michał M
MM
Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland.
Przybylski
Grzegorz K
GK
Smoczkiewicz
Paweł
P
Płotek
Włodzimierz
W
Kowalczyk
Dariusz W
DW
Nowak
Jerzy S
JS
D016694:Gene Rearrangement, delta-Chain T-Cell Antigen Receptor; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013601:T-Lymphocytes