Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (271K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Nazarenko, I.
Right arrow Articles by Rashtchian, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nazarenko, I.
Right arrow Articles by Rashtchian, A.
Related Collections
Right arrow New restriction enzymes
Right arrow Nucleic acid amplification
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2002, Vol. 30, No. 9 e37
© 2002 Oxford University Press

Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore

Irina Nazarenko*, Brian Lowe, Marlene Darfler, Pranvera Ikonomi, David Schuster and Ayoub Rashtchian

Invitrogen Corporation, 1620 Faraday Avenue, Carlsbad, CA 92008, USA

Multiplex quantitative PCR based on novel design of fluorescent primers is described. Fluorogenic primers are labeled with a single fluorophore on a base close to the 3' end with no quencher required. A tail of 5–7 nt is added to the 5' end of the primer to form a blunt-end hairpin when the primer is not incorporated into a PCR product. This design provides a low initial fluorescence of the primers that increases up to 8-fold upon formation of the PCR product. The hairpin oligonucleotides ({Delta}G from –1.6 to –5.8 kcal/mol) may be as efficient as linear primers and provide additional specificity to the PCR by preventing primer-dimers and mispriming. Multiple fluorogenic primers were designed by specialized software and used for real-time quantitation of c-myc and IL-4 cDNAs in the presence of reference genes such as ß-actin, GAPDH and 18S rRNA. Targets of 10–107 copies were detected with precision in PCR using FAM-labeled primers for variable genes and JOE-labeled primers for the reference genes. This method was also used to detect single nucleotide polymorphism of the human retinal degeneration gene by allele-specific PCR with end-point detection using a fluorescent plate reader or a UV-transilluminator. We conclude that fluorogenic mono-labeled primers are an efficient and cost-effective alternative to FRET-labeled oligonucleotides.

* To whom correspondence should be addressed at present address: 708 Quince Orchard Road, Gaithersburg, MD 20878, USA. Tel: +1 301 987 1711; Fax: +1 301 987 1717; Email: inazarenko{at}metrigenix.com


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Smerdel-Ramoya, S. Zanotti, V. Deregowski, and E. Canalis
Connective Tissue Growth Factor Enhances Osteoblastogenesis in Vitro
J. Biol. Chem., August 15, 2008; 283(33): 22690 - 22699.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
J. Nordgren, F. Bucardo, O. Dienus, L. Svensson, and P.-E. Lindgren
Novel Light-Upon-Extension Real-Time PCR Assays for Detection and Quantification of Genogroup I and II Noroviruses in Clinical Specimens
J. Clin. Microbiol., January 1, 2008; 46(1): 164 - 170.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
C. Bonanno, E. Shehi, D. Adlerstein, and G. M. Makrigiorgos
MS-FLAG, a Novel Real-Time Signal Generation Method for Methylation-Specific PCR
Clin. Chem., December 1, 2007; 53(12): 2119 - 2127.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Gazzerro, A. Smerdel-Ramoya, S. Zanotti, L. Stadmeyer, D. Durant, A. N. Economides, and E. Canalis
Conditional Deletion of Gremlin Causes a Transient Increase in Bone Formation and Bone Mass
J. Biol. Chem., October 26, 2007; 282(43): 31549 - 31557.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. Huang, J. Salituro, N. Tang, K.-C. Luk, J. Hackett Jr, P. Swanson, G. Cloherty, W.-B. Mak, J. Robinson, and K. Abravaya
Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR
Nucleic Acids Res., August 13, 2007; 35(16): e101 - e101.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Rydziel, L. Stadmeyer, S. Zanotti, D. Durant, A. Smerdel-Ramoya, and E. Canalis
Nephroblastoma Overexpressed (Nov) Inhibits Osteoblastogenesis and Causes Osteopenia
J. Biol. Chem., July 6, 2007; 282(27): 19762 - 19772.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
J. L. Balcazar, D. Vendrell, I. de Blas, I. Ruiz-Zarzuela, O. Girones, and J. L. Muzquiz
Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers
J. Med. Microbiol., March 1, 2007; 56(3): 323 - 328.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
D. M. Tracz, P. G. Backhouse, A. B. Olson, J. K. McCrea, J. A. Walsh, L.-K. Ng, and M. W. Gilmour
Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus
J. Med. Microbiol., January 1, 2007; 56(1): 56 - 65.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
E. P. Price, F. Huygens, and P. M. Giffard
Fingerprinting of Campylobacter jejuni by Using Resolution-Optimized Binary Gene Targets Derived from Comparative Genome Hybridization Studies
Appl. Envir. Microbiol., December 1, 2006; 72(12): 7793 - 7803.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
J. Martinez-Lopez, P. Martinez-Sanchez, R. Garcia-Sanz, M. E. Sarasquete, R. Ayala, M. Gonzalez, J. M. Bautista, D. Gonzalez, J. S. Miguel, G. Garcia-Effron, et al.
Application of Self-Quenched JH Consensus Primers for Real-Time Quantitative PCR of IGH Gene to Minimal Residual Disease Evaluation in Multiple Myeloma
J. Mol. Diagn., July 1, 2006; 8(3): 364 - 370.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
D. M. Tracz, H. Tabor, M. Jerome, L.-K. Ng, and M. W. Gilmour
Genetic Determinants and Polymorphisms Specific for Human-Adapted Serovars of Salmonella enterica That Cause Enteric Fever.
J. Clin. Microbiol., June 1, 2006; 44(6): 2007 - 2018.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
J. Li, F. Wang, H. Mamon, M. H. Kulke, L. Harris, E. Maher, L. Wang, and G. M. Makrigiorgos
Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples
Clin. Chem., April 1, 2006; 52(4): 624 - 633.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
V. Deregowski, E. Gazzerro, L. Priest, S. Rydziel, and E. Canalis
Notch 1 Overexpression Inhibits Osteoblastogenesis by Suppressing Wnt/beta-Catenin but Not Bone Morphogenetic Protein Signaling
J. Biol. Chem., March 10, 2006; 281(10): 6203 - 6210.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Microbiol.Home page
M. W. Gilmour, D. M. Tracz, A. K. Andrysiak, C. G. Clark, S. Tyson, A. Severini, and L.-K. Ng
Use of the espZ Gene Encoded in the Locus of Enterocyte Effacement for Molecular Typing of Shiga Toxin-Producing Escherichia coli
J. Clin. Microbiol., February 1, 2006; 44(2): 449 - 458.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
D. R. Christensen, L. J. Hartman, B. M. Loveless, M. S. Frye, M. A. Shipley, D. L. Bridge, M. J. Richards, R. S. Kaplan, J. Garrison, C. D. Baldwin, et al.
Detection of Biological Threat Agents by Real-Time PCR: Comparison of Assay Performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler Platforms
Clin. Chem., January 1, 2006; 52(1): 141 - 145.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
R. A.F. Hegazi, K. N. Rao, A. Mayle, A. R. Sepulveda, L. E. Otterbein, and S. E. Plevy
Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway
J. Exp. Med., December 19, 2005; 202(12): 1703 - 1713.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. Kulka, R. Dery, D. Nahirney, M. Duszyk, and A. D. Befus
Differential Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by Interferon {gamma} in Mast Cells and Epithelial Cells
J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 563 - 570.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
D. V. Lim, J. M. Simpson, E. A. Kearns, and M. F. Kramer
Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare
Clin. Microbiol. Rev., October 1, 2005; 18(4): 583 - 607.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
Y. Shinji, T. Tsukui, A. Tatsuguchi, K. Shinoki, M. Kusunoki, K. Suzuki, T. Hiratsuka, K. Wada, S. Futagami, K. Miyake, et al.
Induced microsomal PGE synthase-1 is involved in cyclooxygenase-2-dependent PGE2 production in gastric fibroblasts
Am J Physiol Gastrointest Liver Physiol, February 1, 2005; 288(2): G308 - G315.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. L. Haltiner, T. D. Mitchell, and R. B. S. Harris
Leptin action is modified by an interaction between dietary fat content and ambient temperature
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1250 - R1255.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
M. Varma-Basil, H. El-Hajj, S. A.E. Marras, M. H. Hazbon, J. M. Mann, N. D. Connell, F. R. Kramer, and D. Alland
Molecular Beacons for Multiplex Detection of Four Bacterial Bioterrorism Agents
Clin. Chem., June 1, 2004; 50(6): 1060 - 1062.
[Full Text] [PDF]

Home page
 JCOHome page
H. Endoh, S. Tomida, Y. Yatabe, H. Konishi, H. Osada, K. Tajima, H. Kuwano, T. Takahashi, and T. Mitsudomi
Prognostic Model of Pulmonary Adenocarcinoma by Expression Profiling of Eight Genes As Determined by Quantitative Real-Time Reverse Transcriptase Polymerase Chain Reaction
J. Clin. Oncol., March 1, 2004; 22(5): 811 - 819.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
J.-F. Mercier, G. W. Slater, and P. Mayer
Solid Phase DNA Amplification: A Simple Monte Carlo Lattice Model
Biophys. J., October 1, 2003; 85(4): 2075 - 2086.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
I. Nazarenko, R. Pires, B. Lowe, M. Obaidy, and A. Rashtchian
Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes
Nucleic Acids Res., May 1, 2002; 30(9): 2089 - 2195.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.