Primer sets facilitate study of DNA mismatch repair
Stratagene Cloning Systems
Stratagene has designed RT-PCR primer sets from the cDNA sequences of mismatch repair genes. These genes code for specialized proteins that participate in correcting areas of base mispairing in DNA. We have assembled primer pairs specific for human (hMSH2, hMLH1, hGTBP, hPMS1 and hPMS2) and mouse (mMSH2, mGTBP, mPMS2 and Rep-3) mismatch repair genes. The primer pairs can be used to detect mismatch repair gene transcripts by the reverse transcriptase polymerase chain reaction (RT-PCR). The RT-PCR primer sets for mismatch repair may also be used as positive controls for RT-PCR experiments unrelated to mismatch repair.
Mismatched base pairs in DNA can form in vivo through processes such as replication errors, heteroduplex formation, deamination of 5-methylcytosine and the presence of DNA adducts.1 Cells cope with mismatches by enlisting specialized proteins that can recognize, excise and correct mismatched bases. This process is known as DNA mismatch repair. In addition to correcting single base mispairing, mismatch repair pathways have been suspected to play a role in controlling microsatellite instability in proto-oncogenes2 and in modulating adaptive mutation in nondividing cells.3
The characterization of the E. coli MutHLS mismatch repair system4 led to the identification of an analogous eukaryotic system in Saccharomyces cerevisiae. Proteins in this system include a yeast homologue of MutS, called MSH2,5 and two yeast homologues of MutL, called MLH16 and PMS1.7 Discovery of the human mismatch repair homologues soon followed with the identification of the genes for the hMSH2,8 hMLH1,9 hPMS1,10 hPMS211 and hGTBP12 proteins. Some mouse analogs to previously characterized mismatch repair genes have also been identified. These genes include mMSH2,13 mPMS2 14 and GTMBP 15 (mGTBP). Rep-3 in mice is similar to the MutS gene but shares the greatest homology to yeast MSH3.16
Rapid advances in the field of DNA repair have begun to elucidate the precise role of human mismatch repair proteins.17 In the current model, hMSH2 and hGTBP form a dimer, which recognizes the mismatch and binds to it. Another complex formed by hPMS2 and hMLH1 subsequently recognizes and binds to the DNA-bound, hMSH2-hGTBP complex. Other proteins are then recruited to complete the processes of incision and repair. The human hPMS2 protein performs the analogous function of the PMS1 protein in yeast. The biochemical function of the hPMS1 protein is unclear; however, mutations in the hPMS1 gene, as well as hMSH2, hMLH1 and hPMS2 genes, have been associated with hereditary nonpolyposis colon cancer, a condition marked by microsatellite instability.
Stratagenes RT-PCR primer sets for mismatch repair are designed to amplify cDNA segments representing transcripts of currently characterized mismatch repair genes in humans and mice. Because the RT-PCR primer sets amplify regions that span intron-exon boundaries, amplification using genomic DNA templates produces either no product at all or one that can easily be distinguished from the RT-PCR product by gel electrophoresis. The primer sets can be used in conjunction with quantitative RT-PCR techniques to study gene expression in areas such as genetic toxicology, where changes in the regulation of these genes may be indicative of the genotoxicity of a substance or its biologically activated form. The primer sets may also be used to quickly amplify a region of the featured genes for later use as a probe in other analytical techniques, such as Northern blot hybridization, nuclease protection assays or in situ hybridization. In addition, these probes might be useful for screening cDNA libraries for gene homologues in related species. Because the mismatch repair genes serve an important function in maintaining the genetic integrity of the cell, they are likely to be expressed in detectable levels in most cell types. In this regard, they could be classified as housekeeping genes. Thus, the RT-PCR primer sets could be used as positive controls for cDNA templates in RT-PCR studies unrelated to mismatch repair.
The human RT-PCR primer sets for mismatch repair were used in amplification reactions containing either cDNA template from the human promyelocytic cell line (HL60) or human genomic DNA template (figure 1). Total RNA from the HL60 cell line was isolated using Stratagenes RNA Isolation Kit and quantitated by measuring absorbance at 260 nm. First-strand cDNA was synthesized in a 50-l volume using 5 g of total RNA and Stratagenes RT-PCR Kit according to the recommended protocol. Either 1 to 2 l of the cDNA or 25 to 50 ng of the human genomic DNA was used in 50-l PCR reactions using RoboCycler temperature cyclers. Each reaction was performed in 1X Taq polymerase buffer, 250 M of each dNTP, 10 pmol of each primer and 2.5 U of Taq2000TM DNA polymerase. The temperature cycling profile was as follows: One cycle at 95C for 3 minutes; 30 cycles of 95C for 30 seconds, TA (the recommended annealing temperature for each primer set) for 1 minute and 72C for 1 minute; one cycle at 72C for 10 minutes. Of each PCR reaction, 10 l was run on a 2% agarose, Tris-acetate gel stained with ethidium bromide.
The expected amplification products from human cDNA ranged from 515 to 876 bp. The primer set for hGTBP produced a significant quantity of PCR product as a result of amplification from genomic template, resulting in a 3.5- to 4-kb band. Since this band migrated more slowly than the 515-bp band produced from amplification of cDNA, it is unlikely to cause problems with RT-PCR analysis of cDNA preparations contaminated with genomic DNA. None of the other primer sets resulted in detectable PCR product when amplifying from human genomic DNA template.
A similar experiment contrasted results using the mouse RT-PCR primer sets to amplify either mouse liver cDNA or mouse genomic DNA templates (figure 2). The expected mouse cDNA-derived products in this group ranged from 504 to 926 bp. None of the mouse primer sets amplified a visible product from genomic mouse DNA template when reactions were performed at the recommended annealing temperature.
Characterization of mammalian DNA repair enzymes is a rapidly advancing field. To meet the needs of researchers studying these pathways, Stratagene has developed RT-PCR primers sets for the newly identified genes involved in mammalian DNA mismatch repair. These primers sets amplify regions that span intron-exon boundaries, minimizing the effects of amplification from contaminating genomic DNA template. Each primer set includes resource information for the derivation of the primer set.
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