Navigation Links
RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes

Primer sets facilitate study of DNA mismatch repair


Mark Dycaico
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.

RT-PCR Primer Sets for Mismatch Repair

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.

PCR Amplifications Using Primer Sets

figure 1

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.

figure 2

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.

Conclusions

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.

REFERENCES
  1. Freidberg, E.C., et al. (1995) In DNA Repair and Mutagenesis, pp.367-368. ASM Press, Washington, D.C.

  2. Karran, P. (1996) Semin. Cancer Biol. 7: 15-24.

  3. Longerich, S., et al. (1995) Proc. Natl. Acad. Sci. USA 92: 12017-12020.

  4. Modrich, P. (1991) Annu. Rev. Genet. 25: 229-253.

  5. Reenan, R.S.G., and Kolodner, R.D. (1992) Genetics 132: 963-973.

  6. Strand, M., et al. (1993) Nature 365: 274-276.

  7. Kramer, W., et al. (1989) J. Bacteriol. 171: 5339-5346.

  8. Fishel, R., et al. (1993) Cell 75: 1027-1038.

  9. Bronner, C.E., et al. (1994) Nature 368: 258-261.

  10. Papadopoulos, N., et al. (1994) Science 263: 1625-1629.

  11. Nicolaides, N.C., et al. (1994) Nature 371: 75-80.

  12. Palombo, F., et al. (1995) Science 268: 1912-1914.

  13. Varlet, I., et al. (1994) Nucleic Acids Res. 22: 5723-5728.

  14. Baker, S.M., et al. (1995) Cell 82: 309-319.

  15. Corradi, A., et al. (1996) Unpublished, from Genbank accession number U42190.

  16. Lui, K., et al. (1994) Gene 147: 169-177.

  17. Kolodner, R.D. (1996) Genes and Devel. 10: 1433-1442.


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. Brilliant Core Reagent Kits for QPCR and QRT-PCR
3. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
4. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
5. Performance Comparisons of Commercial RT-PCR Systems
6. RT-PCR Primers for the Study of Apoptosis
7. cMaster RTplusPCR System for increased sensitivity and product length in RT-PCR
8. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
9. Optimized Dengue RT-PCR
10. QuantiTect SYBR Green RT-PCR Kit
11. C. therm. Polymerase One-Step RT-PCR System
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: PCR Primer Sets for Human and Mouse Mismatch Repair Genes

(Date:6/1/2015)... , June 1, 2015 GenomeDx ... genomic analysis platform, successfully classified various subtypes ... expression of certain biomarkers, including one type ... this unique genomic signature has potential as ... neoadjuvant chemotherapy in patients with muscle-invasive bladder ...
(Date:6/1/2015)... - BIOREM Inc. (TSX-V: BRM) ("Biorem" or "the Company") today announced results ... 2015 first quarter financial statements and MD&A have been filed on ... March 31,(in CDN$,000 except per share data) , 2015 ... Gross profit , 1,531 , 329 Ebitda* , ... 528 , (531) Basic earnings (loss) per share ...
(Date:6/1/2015)... Research and Markets ( ... "Global PDT Machine Industry Report 2014" ...      (Logo: http://photos.prnewswire.com/prnh/20130307/600769) , , ,The Global ... and in-depth study on the current state of ... basic overview of the industry including definitions, classifications, ...
(Date:6/1/2015)... 01, 2015 Regis Technologies, Inc. ... of chiral stationary phases (CSPs) for analytical and ... current and presenting solutions for new clients, Regis ... This valuable addition to its core expertise in ... that can tackle projects for the scale clients ...
Breaking Biology Technology:GenomeDx's Decipher Platform Validates Genomic Signature that may Predict Resistance to Chemotherapy in Bladder Cancer Patients in Study Presented at ASCO 2015 Annual Meeting 2GenomeDx's Decipher Platform Validates Genomic Signature that may Predict Resistance to Chemotherapy in Bladder Cancer Patients in Study Presented at ASCO 2015 Annual Meeting 3GenomeDx's Decipher Platform Validates Genomic Signature that may Predict Resistance to Chemotherapy in Bladder Cancer Patients in Study Presented at ASCO 2015 Annual Meeting 4Biorem Reports Increased Revenues and Earnings for First Quarter 2Biorem Reports Increased Revenues and Earnings for First Quarter 3Global PDT Machine Industry Report 2014 2Regis Technologies Offers Chiral Chromatography Service 2Regis Technologies Offers Chiral Chromatography Service 3
... workforce reduction, NEW YORK, April 4, 2008 ... that it is implementing a,strategic restructuring plan to ... efforts. The plan, which was prompted by the ... clinical trial of Sulonex(TM),(sulodexide) for the treatment of ...
... Pharmaceuticals,Inc. (NASDAQ: ANPI , TSX: ANP), a ... that the first New Zealand patient,was treated with ... December 2007. A second patient was treated in ... drug-eluting stent for peripheral,arterial disease (PAD) and was ...
... held at the Society for Biomolecular Sciences 14th ... ... Ltd., an,international provider of microfluidic systems in the emerging field of,nano-lifesciences, ... enables researchers to execute physiologically,relevant flow experiments evaluating the adhesion of ...
Cached Biology Technology:Keryx Biopharmaceuticals Announces Implementation of Strategic Restructuring Plan 2Keryx Biopharmaceuticals Announces Implementation of Strategic Restructuring Plan 3Keryx Biopharmaceuticals Announces Implementation of Strategic Restructuring Plan 4Angiotech announces commercial launch of Cook Medical's Zilver(R) PTX(TM) drug-eluting peripheral stent in New Zealand 2Angiotech announces commercial launch of Cook Medical's Zilver(R) PTX(TM) drug-eluting peripheral stent in New Zealand 3Angiotech announces commercial launch of Cook Medical's Zilver(R) PTX(TM) drug-eluting peripheral stent in New Zealand 4Cellix Ltd. Launches VenaEC Biochip for Flow-based Cell-adhesion Assays 2
(Date:5/19/2015)... YORK , May 19, 2015  Technology is ... both internally and in the cloud. Passwords and their ... use of OTP and standards-based specifications such as those ... the outmoded use of passwords presents for BYOD, COPE, ... a unified biometric identity protocol. In ...
(Date:5/11/2015)... 2015  Synaptics Incorporated (NASDAQ: SYNA ), ... announced the appointment of Wajid Ali ... reporting to Rick Bergman , President and ... Officer, Kathleen Bayless , who announced her ... Ali brings extensive financial management expertise to Synaptics, ...
(Date:5/7/2015)... 7, 2015 Fingerprint Cards ... and FPC1035, FPC,s smallest touch fingerprint sensors to ... considered for integration on the backside of the ... increased possibilities to integrate touch fingerprint sensors in ... improves possibilities for module manufacturers to customize the ...
Breaking Biology News(10 mins):HYPR Corp. Biometric Security Integration Kit Shields Applications and Devices Across the Internet of Things 2HYPR Corp. Biometric Security Integration Kit Shields Applications and Devices Across the Internet of Things 3Synaptics Appoints Wajid Ali as Senior Vice President and Chief Financial Officer 2FPC Introduces its Smallest Touch Fingerprint Sensors to Date 2
... a salt sea without an outlet, lies over 400 ... content because it increases their buoyancy. "For scientists, ... provides a diachronic view of its climate past," says ... Mineralogy and Paleontology at the University of Bonn. ...
... has developed a framework to evaluate the risk of ... paper based on this work: A framework for assessing ... been published in the Malaria Journal today. ... drug, so it is important when developing new medicines ...
... effort to document environmental change by monitoring and ... The country,s inclusion made it the newest ... Drilling Program (IODP). IODP scientists conduct research ... of the Earth through drilling, coring, monitoring and ...
Cached Biology News:Drastic desertification 2Drastic desertification 3MMV develops framework to assess risk of resistance for antimalarial compounds 2Brazil joins international marine research effort 2
... Introducing SilverSNAP Stain for Mass Spectrometry ... proteome, mass spectrometry (MS) has emerged as ... Silver staining of 2-D gels is now ... of procedures that leads ultimately to identification ...
... PCR System is the only PCR system ... and every time. It provides accuracy, consistency, ... source or the property of the DNA ... Enzyme Mix, a unique blend of thermostable ...
... cleaning agent for removing RNase ... and pipettors. It is ... contamination from microcentrifuge tubes without ... Zap (R) is ...
A set of five proteins which can be used to calibrate a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or Electrospray Ionization (ESI) mass spectrometer....
Biology Products: