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bin (G20210A), Factor V (G169I), and methylenetetrahydrofolate reductase (C677T) by real-time fluorescence PCR with the LightCycler. Clin. Chem. 45, 694 – 696. Frosst, P., Blom, H. J., Milos, R., Goyette, P., Sheppard, C. A., Matthews, R. G., Boers, G. J., den Heijer, M., Kluijtmans, L. A., and van den Heuvel, L. P. (1995) A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 10, 111–113. Kimura, H., Gejyo, F., Suzuki, S., and Miyazaki, R. (2000) The C677T methylenetetrahydrofolate reductase gene mutation in hemodialysis patients. J. Am. Soc. Nephrol. 11, 885– 893. van der Put, N. M., Gabreels, F., Stevens, E. M., Smeitink, J. A., Trijbels, F. J., Eskes, T. K., van den Heuvel, L. P., and Blom, H. J. (1998) A second common mutation in the methylenetetrahydrofolate reductase gene: An additional risk factor for neuraltube defect? Am. J. Hum. Genet. 62, 1044 –1051. Weisberg, I. S., Jacques, P. F., Selhub, J., Bostom, A. G., Chen, Z., Curtis Ellison, R., Eckfeldt, J. H., and Rozen, R. (2001) The 1298A f C polymorphism in methylenetetrahydrofolate reductase (MTHFR): In vitro expression and association with homocysteine. Atherosclerosis 156, 409 – 415. Sanger, G., Goldstein C., and van Miltenburg, R. (1999) Detection of multiple reporter dyes in real-time, on-line PCR analysis with the LightCycler system. Roche Biochemica No. 2, pp. 7–11. LightCycler-Red 705-phosphoramidite. (1999) Roche Biochemica No. 2, p. 19. Choi, S. W., and Masen, J. B. (2000) Folate and carcinogenesis: An integrated scheme. J. Nutr. 130, 129 –132.
Modifying Differential Display Polymerase Chain Reaction to Detect Relative Changes in Gene Expression Profiles Daya G. Ranamukhaarachchi, Mangalathu S. Rajeevan, 1 Suzanne D. Vernon, and Elizabeth R. Unger Viral Exanthems and Herpesvirus Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, 1600 Clifton Road, Atlanta, Georgia 30333 Received December 21, 2001; published online June 11, 2002
Differential display PCR (DD-PCR) 2 is an increasingly important tool for gene expression profiling studies. Since the introduction of DD-PCR numerous advances have been made in minimizing false positives and in adapting the technology to multicolor fluorescence detection (1, 2). However, DD-PCR band intensities do not accurately reflect expression levels since 1 To whom correspondence and reprint request should be addressed. Fax: 404-639-3540. E-mail: [email protected]
2 Abbreviations used: DD-PCR, differential display PCR; RAPPCR, RNA arbitrarily primed PCR; RT, reverse transcription; TMR, tetramethylrhodamine.
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100-fold differences in the amount of input RNA give comparable results (3, 4). Therefore, current DD-PCR conditions would fail to detect differentially expressed genes within the 10-fold range that microarray studies have identified as the most common level of regulation (5, 6). In this study we evaluate the impact of changing the amount of input cDNA and the number of high-stringency PCR cycles on the ability of fluorescent DD-PCR to produce complex expression profiles with band intensities that reflect known 2- to 10-fold dilutions of target. We found that for most primer combinations, 4-fold less cDNA and high-stringency PCR cycles reduced to 25 produced reproducible complex band patterns with intensities that reflected 2- to 10-fold differences in expression levels. Real-time quantitative PCR confirmed 90% of differentially expressed genes detected by this modified DD-PCR. These simple changes can be used to make fluorescence DD-PCR more quantitative and may be applied to related differential display techniques such as RNA arbitrarily primed PCR (RAP-PCR (7)). MATERIALS AND METHODS
Fluorescence DD-PCR The cervical cancer cell line Caski was grown as recommended by the American Type Culture Collection (Rockville, MD). Methods for RNA extraction, removal of contaminating DNA, and determination of RNA quantity and quality were reported earlier (5). Reverse transcription (RT) reaction (20 l) was performed in a GeneAmp 9600 thermocycler (PE Applied Biosystems, Foster City, CA) following the protocol of the Hieroglyph mRNA profile kit (Beckman Coulter Corp., Foster City, CA) with 0.2 g total RNA, 0.2 M oligo(dT) anchored primer, 40 units Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA), and 20 units RNasin (Promega, Madison, WI). Tetramethylrhodamine (TMR)-labeled oligo(dT) anchored primers (Fluoro DD-Adaptor kit; Beckman Coulter) together with arbitrary primers (Hieroglyph mRNA profile kit; Beckman Coulter) were used to generate fluorescent PCR products. Two-fold serial dilutions (1:1 to 1:128) of the RT product were prepared in water and 1 l was used in the 10-l PCR mixture. The number of highstringency cycles was varied from 15 to 30 using a GenAmp 9600 thermocycler (PE Applied Biosystems). The remaining reagents and reaction conditions were as specified in the Fluoro DD-PCR manual (Beckman Coulter). Each PCR was performed in duplicate to verify the reproducibility of bands. PCR products (4 l) were fractionated at 2700 V/100 W for 5 h using 5.6% clear denaturing HR-1000 gel (Beckman Coulter) and Genomyx LR sequencing gel electrophoresis apparatus (Beckman Coulter). TMR-labeled DNA size standards (300 –2000 bp) were obtained from Beckman Coulter.
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Gels were scanned by the Genomyx SC fluorescence scanner (Beckman Coulter) and images were saved as TIFF files in Adobe PhotoShop 5.0 (Adobe Systems, Inc., San Jose, CA). Image Analysis Images were analyzed visually. We compared the band number and intensity in the cDNA dilutions series. The goal was to determine conditions that maintained a complex reproducible band pattern with band intensities that changed in proportion to the dilution of the cDNA. RESULTS AND DISCUSSION
Studies that identified the failure of differential display techniques to detect 100-fold differences in gene expression used 40 low-stringency cycles and detected PCR products by incorporation of radioactive nucleotides and autoradiography (3, 4). Here, we first evaluated if standard fluorescence DD-PCR based on 30 high-stringency cycles and detection of end-labeled PCR products also showed a lack of quantitation by performing PCR on 2-fold serial dilutions of the cDNA template (1- to-128-fold dilutions). As shown in Fig. 1, nearly identical and complex banding patterns were obtained for dilutions up to 8-fold using the recommended amount of input cDNA. Higher cDNA dilutions (16- to 128-fold) resulted in reduced band complexity and substantially reduced reproducibility between duplicate reactions. The near equivalence of band intensity for the 8-fold dilutions indicates that under these conditions, DD-PCR would not be able to distinguish less than a 16-fold change in gene expression. The failure to detect a difference in band intensity cannot be attributed to the scanner and detection system, since a 2-fold dilution of the molecular weight markers can be readily distinguished (marker DNA; Fig. 1). Similar experiments were done with three Taq DNA polymerases, viz., Platinum Taq DNA polymerase (Invitrogen) and AmpliTaq and AmpliTaq Gold DNA polymerases (PE Applied Biosystems). Both Platinum Taq and AmpliTaq produced similar and complex gene expression profiles, whereas AmpliTaq Gold resulted in profiles with reduced complexity with all amounts of input cDNA. Results from these and previous studies taken together imply that the failure of DD-PCR to detect quantitative differences in gene expression is not dependent on the sources of RNA, primer length, PCR product labeling methods (nucleotide incorporation vs end-labeling), detection (fluorescence vs radioactive), or Taq DNA polymerases (AmpliTaq vs Platinum Taq). Compared to radioactive DD-PCR with 40 cycles, fluorescence DD-PCR used only 30 cycles; still expression
FIG. 1. Analysis of the ability of standard fluorescence DD-PCR conditions to detect differences in input cDNA. Two-fold Caski cDNA dilution series were tested under standard conditions of fluorescence DD-PCR (30 high-stringency cycles). 1X–128X indicate dilutions of cDNA prior to PCR amplification or dilutions of unamplified marker DNA. Note the similar intensities of bands for dilutions up to 8-fold. Higher dilutions (⬎16-fold) often showed lack of band reproducibility (boxed area). Results shown were obtained with AP5/ARP2 primer combination (Hieroglyph mRNA profile kit; Beckman Coulter) and Platinum Taq DNA polymerase. AmpliTaq gave similar results.
profiles with the standard fluorescence DD-PCR were similar up to 8-fold dilutions of input cDNA. We reasoned that the ability of DD-PCR to detect the range of differences in gene expression levels (⬍10-fold) most commonly observed in microarray and kinetic PCR studies (5, 6) would depend primarily on adjusting the
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amount of input cDNA and number of PCR cycles. Since DD-PCR profiles are generated by hundreds of combinations of anchored and arbitrary primers, the optimized conditions should ideally be applicable to a large number of primer combinations. Based on these considerations, we first analyzed expression profiles generated with 2-fold serial dilutions of input cDNA and various numbers of PCR cycles using a single primer combination. As shown in Fig. 2A, compared with the standard 30 cycles, 25 cycles resulted in the same complex band pattern but with visual differences in intensity for cDNA dilutions between 1- and 16-fold. Further reduction in cycle number to 20 resulted in weak bands and a substantial reduction in band complexity. Fifteen cycles of PCR did not generate any bands (data not shown). These experiments suggested that the amount of input cDNA could be lowered at least four-fold without loss of band complexity and that reduced cDNA template along with reduced high-stringency PCR cycles was essential to permit band intensities to visually reflect 2- to 8-fold differences in template. The reproducibility of DD-PCR with 25 high-stringency PCR cycles using four-fold less cDNA was tested by comparing results obtained from two independent RT reactions from the same total RNA preparation. As shown in Fig. 2B, the fingerprint generated with the reduced template and optimized cycle number gave nearly identical results, indicating the high reproducibility of these conditions. We have applied these conditions using 29 different primer combinations to compare the expression profiles of cervical keratinocytes grown under differentiating or nondifferentiating conditions. Approximately, 85% (24 of 29) of the primer combinations yielded profiles with complex fingerprints, 50 –100 bands that ranged from 300 to 1800 bp. From a total of 191 bands identified as differentially expressed based on visual differences in band intensity (2- to 4-fold, 5- to 10-fold, ⬎10-fold, and unique), we selected a representative set of 13 for validation of the actual expression level using real-time (kinetic) PCR (8). A very high validation rate (12/13; ⬎90%) was shown for these bands. We did note that some primer combinations (5/29; 15%) required the full 30 cycles of high-stringency PCR to generate a complex banding pattern. The problem of poor band complexity seen with a few primer pairs may be due to low priming efficiency. As a general approach, we recommend screening DD-PCR profiles with as many primer combinations as possible using the optimal conditions identified in this report. Input cDNA and PCR cycle numbers may be increased either alone or in combination subsequently for those primer combinations with low priming efficiency in the first screen. Any alteration in reaction conditions that affects PCR efficiency could influence the optimum cycle number. Therefore any alteration in
FIG. 2. (A) Impact of cycle number on the ability of DD-PCR to reflect differences in input cDNA. Expression profile of the Caski cell line was generated as in Fig. 1 except that cycle number was varied from 30 to 15. Reducing high-stringency PCR cycles from 30 to 25 allowed band intensity to reflect Caski cDNA dilutions without reduction in band complexity. Further reduction in cycle number to 20 compromised the complexity. Fifteen cycles of PCR did not generate any bands (data not shown). 1X, 4X, and 16X indicate dilutions of cDNA prior to PCR amplification. (B) Reproducibility of new conditions for fluorescent DD-PCR. Duplicate RT reactions (RT1 and RT2) from the same Caski total RNA were analyzed in duplicate PCRs under optimized conditions (4-fold dilution of cDNA, 25 high-stringency PCR cycles). Duplicate PCR with RT1 product (lanes 1, 2). Duplicate PCR with RT2 product (lanes 3, 4). Marker (lane M). Expression profiles in lanes 1 through 4 are nearly identical.
DD-PCR conditions should be tested empirically to determine the impact on band intensity, complexity, reproducibility, and ability to reflect known dilutions of template. Testing dilution series of cDNA with varying cycle number is an effective tool for this optimization (limited to one or two gels with one sample and two or three primer combinations) and is recommended for initial experiments with differential display techniques such as DD-PCR and RAP-PCR.
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Current DD-PCR protocols using both radioactivity and fluorescence detection require at least 4 – 8 g of total RNA per sample for complete gene expression profiling with additional material required for validation. As an added benefit, our protocol uses four-fold less RT product in each PCR and enables genome-wide expression profiles to be performed with as little as 1 g of total RNA. This is a significant advantage for gene expression profiling in molecular epidemiological studies that rely on samples of limited quantity. Acknowledgments. The authors acknowledge the technical assistance of Navoarath Taysavang and Daisy Lee for providing Caski cells.
REFERENCES 1. Belomon, J. W., and Jurecic, R. (2000) Long-distance DD-PCR and cDNA microarrays. Curr. Opin. Microbiol. 3, 316 –321. 2. Cho, Y.-j., Meade, J. D., Walden, J. C., Chen, X., Guo, Z., and Liang, P. (2001) Multicolor fluorescent differential display. BioTechniques 30, 562–572. 3. Liang, P., Averboukh, L., and Pardee, A. B. (1993) Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization. Nucleic Acids Res. 21, 3269 –3275. 4. Bosch, I., Melichar, H., and Pardee, A. B. (2000) Identification of differentially expressed genes from limited amounts of RNA. Nucleic Acids Res. 28, E27. 5. Rajeevan, M. S., Vernon, S. D., Taysavang, N., and Unger, E. R. (2001) Validation of array-based gene expression profiles by realtime (kinetic) RT-PCR. J. Mol. Diagn. 3, 26 –31. 6. Der, S. D., Zhou, A., Williams, B. R. G., and Silverman, R. H. (1998) Identification of genes differentially regulated by interferon ␣, ␤, or ␥ using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95, 15623–15628. 7. Mathieu-Daude, F., Trenkle, T., Welsh, J., Jung, B., Vogt, T., and McCleland, M. (1999) Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction. Methods Enzymol. 303, 309 –324. 8. Rajeevan, M. S., Ranamukhaarachchi, D. G., Vernon, S. D., and Unger, E. R. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display-PCR technologies. Methods 25, 443– 451.
Site-Directed Mutagenesis by the Megaprimer PCR Method: Variations on a Theme for Simultaneous Introduction of Multiple Mutations Sebastiana Angelaccio and Maria Carmela Bonaccorsi di Patti Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Universita` degli Studi di Roma “La Sapienza,” P.le Aldo Moro 5, 00185 Rome, Italy Received January 28, 2002; published online June 11, 2002
Most of the published methods concerning the use of PCR for site-directed mutagenesis deal with the introAnalytical Biochemistry 306, 346 –349 (2002) doi:10.1006/abio.2002.5689 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
duction of a single mutation (1). Methods for the simultaneous introduction of multiple mutations involve the use of thermostable DNA ligase, such as in the ligase chain reaction (2) or in the combined chain reaction (3). Alternatively, a method which uses overlap extension PCR has been reported (4); however, it requires a large number of primers (six) to generate three overlapping DNA fragments. The megaprimer method is widely used to introduce desired mutations into target DNA sequences, by way of two rounds of PCR that utilize two flanking primers and an internal mutagenic primer (5). In the first PCR the mutagenic primer is used together with the appropriate flanking primer; the double-stranded megaprimer which is produced is then purified and used, together with the second flanking primer, as a primer for a second PCR to amplify the mutated DNA. By employing two novel approaches, we have expanded the potential of the megaprimer method in order to simultaneously introduce multiple nucleotide substitutions into different regions of a DNA sequence. The first approach takes advantage of the fact that the megaprimer is double-stranded. As shown in Fig. 1a, a single internal megaprimer is produced with two oligonucleotide primers carrying all the desired mutations (C and D). The megaprimer is purified and used in two separate PCRs with each of the flanking primers A and B to produce two mutated DNA fragments. These two fragments contain an overlapping region corresponding to the megaprimer and they are then used as template in a third PCR with the flanking primers A and B to obtain the full-length mutated DNA. This procedure has allowed us to simultaneously introduce up to six amino acid substitutions in Saccharomyces cerevisiae Fet3 (Table 1). Primers A and B, which carry EcoRI restriction sites (underlined) and anneal at the 5⬘ and 3⬘ ends of Fet3 cDNA, were used as common flanking primers; the mutagenic primers employed to obtain either Fet3 F219I/F277I or Fet3 D278Q/D279-312-315-319-320N mutants are shown in Table 1. The first PCR was performed in 50 l containing 10 ng template plasmid DNA pBSFet3 (6), 10 nmol dNTP, 25 pmol each mutagenic primer, the appropriate buffer, and 2 U Expand High Fidelity DNA polymerase (Roche). Conditions were identical for production of the double mutant and the six-mutant megaprimer and were 25 cycles at 95°C, 1 min; 58°C, 45 s; 72°C, 45 s; and a final extension at 72°C for 10 min. The megaprimers (195 and 141 bp long, respectively) were gel-purified and employed for the second PCR (100 ng) in two separate reactions with each of the flanking primers and 300 ng pBSFet3. PCR conditions were the same as before, except that annealing was performed at 60°C for 1 min and extension time was 1 min. The yield of products I and II of this step may vary, depending on the efficiency with which the two