PL EN


Preferences help
enabled [disable] Abstract
Number of results
2002 | 43 | 2 | 235-244
Article title

Novel competitive PCR methods for quantitation of T-cell receptor delta (TCRD) gene rearrangements

Content
Title variants
Languages of publication
EN
Abstracts
EN
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 Jd1 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.
Discipline
Year
Volume
43
Issue
2
Pages
235-244
Physical description
References
Document Type
ARTICLE
Publication order reference
G. K. PrzybylskI, Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479 Poznan, Poland
YADDA identifier
bwmeta1.element.element-from-psjc-74cf4c23-3bd6-3ae1-aedf-cffd1b5c6f43
Identifiers
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.