Doubling Throughput of a Real-Time PCR

Overview

Journal Sci Rep

Specialty Science

Date 2015 Jul 28

PMID 26213283

Citations 10

Authors

Christian D Ahrberg

Pavel Neuzil

Affiliations

Soon will be listed here.

Abstract

The invention of polymerase chain reaction (PCR) in 1983 revolutionized many areas of science, due to its ability to multiply a number of copies of DNA sequences (known as amplicons). Here we report on a method to double the throughput of quantitative PCR which could be especially useful for PCR-based mass screening. We concurrently amplified two target genes using only single fluorescent dye. A FAM probe labelled olionucleotide was attached to a quencher for one amplicon while the second one was without a probe. The PCR was performed in the presence of the intercalating dye SYBR Green I. We collected the fluorescence amplitude at two points per PCR cycle, at the denaturation and extension steps. The signal at denaturation is related only to the amplicon with the FAM probe while the amplitude at the extension contained information from both amplicons. We thus detected two genes within the same well using a single fluorescent channel. Any commercial real-time PCR systems can use this method doubling the number of detected genes. The method can be used for absolute quantification of DNA using a known concentration of housekeeping gene at one fluorescent channel.

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References

Rajagopal A, Scherer A, Homyk A, Kartalov E . Supercolor coding methods for large-scale multiplexing of biochemical assays. Anal Chem. 2013; 85(16):7629-36. DOI: 10.1021/ac401304t. View

Kim Y, Choi Y, Jeon B, Jin H, Cho S, Lee H . A simple and efficient multiplex PCR assay for the identification of Mycobacterium genus and Mycobacterium tuberculosis complex to the species level. Yonsei Med J. 2013; 54(5):1220-6. PMC: 3743195. DOI: 10.3349/ymj.2013.54.5.1220. View

Safdar M, Abasiyanik M . Simultaneous identification of pork and poultry origins in pet foods by a quick multiplex real-time PCR assay using EvaGreen florescence dye. Appl Biochem Biotechnol. 2013; 171(7):1855-64. DOI: 10.1007/s12010-013-0485-7. View

Moniotte S, Vaerman J, Kockx M, Larrouy D, Langin D, Noirhomme P . Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. J Mol Cell Cardiol. 2001; 33(12):2121-33. DOI: 10.1006/jmcc.2001.1475. View

Nazarenko I, Lowe B, Darfler M, Ikonomi P, Schuster D, Rashtchian A . Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res. 2002; 30(9):e37. PMC: 113860. DOI: 10.1093/nar/30.9.e37. View

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

Ward C, Dempsey M, Ring C, Kempson R, Zhang L, Gor D . Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. J Clin Virol. 2004; 29(3):179-88. PMC: 7128145. DOI: 10.1016/S1386-6532(03)00122-7. View

Saiki R, Scharf S, Faloona F, Mullis K, Horn G, Erlich H . Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985; 230(4732):1350-4. DOI: 10.1126/science.2999980. View

Holland P, Abramson R, Watson R, Gelfand D . Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991; 88(16):7276-80. PMC: 52277. DOI: 10.1073/pnas.88.16.7276. View

10.

Higuchi R, Fockler C, Dollinger G, Watson R . Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y). 1993; 11(9):1026-30. DOI: 10.1038/nbt0993-1026. View

11.

Witham P, Yamashiro C, Livak K, Batt C . A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Appl Environ Microbiol. 1996; 62(4):1347-53. PMC: 167901. DOI: 10.1128/aem.62.4.1347-1353.1996. View

12.

Andrus A, Cox S, Beavers S, Parker A, Anuskiewicz J, Mullah B . High-throughput synthesis of functionalized oligonucleotides. Nucleic Acids Symp Ser. 1997; (37):317-8. View

13.

Bohling S, King T, Wittwer C, Elenitoba-Johnson K . Rapid simultaneous amplification and detection of the MBR/JH chromosomal translocation by fluorescence melting curve analysis. Am J Pathol. 1999; 154(1):97-103. PMC: 1853425. DOI: 10.1016/S0002-9440(10)65255-8. View

14.

Lind K, Stahlberg A, Zoric N, Kubista M . Combining sequence-specific probes and DNA binding dyes in real-time PCR for specific nucleic acid quantification and melting curve analysis. Biotechniques. 2006; 40(3):315-9. DOI: 10.2144/000112101. View

15.

Lo C, Yip S, Cheng P, To T, Lim W, Leung P . One-step rapid reverse transcription-PCR assay for detecting and typing dengue viruses with GC tail and induced fluorescence resonance energy transfer techniques for melting temperature and color multiplexing. Clin Chem. 2007; 53(4):594-9. DOI: 10.1373/clinchem.2006.077446. View

16.

Gudnason H, Dufva M, Bang D, Wolff A . Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 2007; 35(19):e127. PMC: 2095797. DOI: 10.1093/nar/gkm671. View

17.

Cheah E, Malkin J, Free R, Lee S, Perera N, Woltmann G . A two-tube combined TaqMan/SYBR Green assay to identify mycobacteria and detect single global lineage-defining polymorphisms in Mycobacterium tuberculosis. J Mol Diagn. 2010; 12(2):250-6. PMC: 2871733. DOI: 10.2353/jmoldx.2010.090030. View

18.

Huang Q, Zheng L, Zhu Y, Zhang J, Wen H, Huang J . Multicolor combinatorial probe coding for real-time PCR. PLoS One. 2011; 6(1):e16033. PMC: 3021529. DOI: 10.1371/journal.pone.0016033. View

19.

Van Poucke M, Van Zeveren A, Peelman L . [Letter to the editor] Combined FAM-labeled TaqMan probe detection and SYBR green I melting curve analysis in multiprobe qPCR genotyping assays. Biotechniques. 2012; 52(2):81-6. DOI: 10.2144/000113808. View

20.

Tan S, Carr C, Yeoh K, Schofield C, Davies K, Clarke K . Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors. Mol Biol Rep. 2011; 39(4):4857-67. PMC: 3294216. DOI: 10.1007/s11033-011-1281-5. View