Construction Of Heteroduplex DNA And In Vitro Model Of Functional Analysis Of Mismatch Repair As Well As Its Application In The Diffuse Large B Cell Lymphoma

Objective Using bacteriophage M13mp2 and its derivatives to construct two kinds of heteroduplex DNA molecular serving as templates for mismatch repair, with one possessing single base mismatching and the other possessing two bases deletion. Lymphoblast lymphoma cell line TK6 of mismatch repair (MMR) competency and Lovo of mismatch repair deficiency were used as the positive and negative mismatch control, respectively. Then the model for the functional analysis of mismatch repair in vitro has been set up by this heteroduplex DNA and both of these two cell lines. Using this model to measure the mismatch repair ability in one case of clinically/molecular genetically diagnosed hereditary non-polyposis colorectal caner (HNPCC) fulfilling Amsterdam Criteria and one case of sporadic rectal cancer so that its sensitivity and specificity can be evaluated. Then in the diffuse large B cell lymphoma (DLBCL) the same model was used to investigate the correlation between the MMR status and the tumorigenesis of the DLBCL together with other mismatch repair assays.Method The character of specific life history of the M13mp2 decides that every type of M13mp2 has two kinds of DNA, double strand DNA (dsDNA) and single strand DNA (ssDNA) could be used for the construction of heteroduplex. Extraction of the replication form (RF) circular dsDNA from one kind of phage (wild type or TCA) and digestion of it by restriction enzyme was performed. The linear ssDNA through denaturation was obtained. By annealing of this ssDNA with other kind circular ssDNA extracted from other kind of M13mp2 (del (2) or TGA) a heteroduplex DNA has been constructed and was purified from 0.8% agar-rose gel. In this heteroduplex DNA one strand had a premade nick, termed (-) strand with the other strand termed (+) strand. All the bases in this nicked circular dsDNA are matched according toWatson-Crick rule except the mismatch base pair or un-match bases arranged beforehand in the special site. Depending on the introduction of this heteroduplex molecular into E.coli NR9162 (rnutS) lacking MMR ability by electroporation and the cultivation of the transfected E.coli in the indicator plate with x-gal and IPTG three different colored plaques of blue, clear and mixture plaque were seen, which represented the different characteristic phenotypes of the reconstructed clones of heteroduplex DNA.Human lymphoblastoid B-cell lymphoma cell line TK6 with MMR ability and human colorectal carcinoma cell line Lovo with a homozygous deletion in the hMSH2 gene from exon 3 to exon 8 therefore MMR deficient as well as the constructed heteroduplex were supplied for the establishment of a complete in vitro mismatch repair model. Whole protein from these two cell lines was extracted and measured by large T-antigen dependent SV-40 DNA replication ability assay, which was used as the quality control of the protein. The extractions were then incubated with the heteroduplex DNA. Calculation of the repair efficiency according to the rate change of the mixture plaque before and after incubating, and the functional status of the MMR system in these cells was evaluated.Additional extraction of whole protein from one case of HNPCC and one case of sporadic rectal cancer was set to the established in vitro mismatch repair system for evaluation of their MMR functional status. The miscrosatellite analysis was carried out in these two cases using the 5 panels recommended by the HNPCC international co-laborative group.DNA from 25 cases of DLBCL was extracted and microsatellite status of 7 mono nucleotide repeat loci was studied. The immuno-histochemistry staining of PCNA was also processed in the paraffin embedded slides of the same cases. Extraction of the whole protein from these tumor tissues stored in the liquid nitrogen was performed. Only the successful cases with large T-antigen dependent SV-40 DNA replication ability was observed for the MMR ability of mismatch repair in the constructed in vitro model.Results The mixture plaques immerged in the indicator plate with x-gal and IPTG afte...