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

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Masternak M. M., Przybylski G. K., Smoczkiewicz P., Plotek W., Kowalczyk D. W., Nowak J. S.

Journal of Applied Genetics

2002

vol. 43

issue 2

235-244

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.

Familial polyposiscoli inducing mutatios in APC gene in Poland

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Pawlak A. L., Plawski A., Smoczkiewicz P., Kwiatkowska J., Meissner W., Krokiewicz P., West S. P.

vol. 38

issue 1

77-85

Screening for molecular changes within the adenomatous polyposis coli (APC) gene, exons 11 14 and the 5' half of exon 15, encompassing the mutation cluster region within exon 15, was performed in 30 patients with Familial Polyposis Coli (FAP). All patients were studied by heteroduplex analysis (HA) and single strand conformation polymorphism (SSCP) and molecular changes were found in 7 cases. Protein truncation test (PTT) has been performed in 17 cases in which mutations have not been found earlier, and shortening of protein product was noted in 2 cases. In three cases common deletion of 5 bp at codon 1309 and in one 5 bp deletion at codon 1061 were found. In other cases the molecular changes were demonstrated as heteroduplexes in exon 14 (1 patient), in segments E and F (one patient each) of exon 15, and in two cases the heteroduplexes were within the overlapping sequences of segments E/F and F/G of exon 15, respectively. In families where the molecular changes were found by HA, 7 persons at high risk for FAP were found and advised to undergo regular endoscopic examinations. In three persons at risk the transfer of mutation was excluded.

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2 Journal of Applied Genetics

1 2002

1 1997

2 Smoczkiewicz P.

1 Kowalczyk D. W.

1 Krokiewicz P.

1 Kwiatkowska J.

1 Masternak M. M.

1 Meissner W.

1 Nowak J. S.

1 Pawlak A. L.

1 Plawski A.

1 Plotek W.

1 Przybylski G. K.

1 West S. P.

Search results

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

Familial polyposiscoli inducing mutatios in APC gene in Poland