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Site-Directed Mutation and PCR Mimics 209 for samples assayed within a single assay the variation can be kept within acceptable limits. Furthermore, although it is possible, using the pCRII vector, to generate mRNA as an additional control, the low intra assay variation defined in this system allows this simpler procedure to be followed. Interassay and intra-assay variations were similar to those obtained by conventional radioimmunoassays (2,3), suggesting this technique could be robustly applied to clinical diagnostic problems, such as the determination of viral load for either RNA or DNA viruses (omitting the RT step for the latter). The sensitivity of this technique is such that low copy number genes could be assayed in 100 s or at most a few thousand cells and can be applied to patient tissue samples and small cell cultures. In addition, by allowing absolute concentrations to be determined, this assay will facilitate comparisons between laboratories previously hampered by semiquantitative approaches to PCR (4,5), and this precision is maintained even over most applications of fluorescence real-time PCR (6). 4. Notes 1. The cDNA sequence for human cAMP-dependent protein kinase subunit was retrieved from the Gembl database (accession no. M33336; 7). Using this sequence, primers were designed that amplified bases 159 to 589. This 430-bp fragment codes entirely for mRNA, which is subsequently translated into protein. 2. The mimic is inserted in the 3900-bp pCRII vector by calculating the length of the vector containing the insert and dividing this by the length of the insert the molecular weight fraction made up by the insert is calculated and the Mwt corrected to reflect that of the PCR insert only. The molar equivalent for RI alpha mRNA added to the assay system was calculated as follows: 1 pg RI alpha plasmid is equivalent to 0.34 fmols RI alpha mRNA. 3. The PCR mimic was constructed as described elsewhere. Using site-directed mutagenesis, a single bp change was introduced within the RI alpha PCR product to introduce a EcoRV restriction site. For researchers wishing to evaluate this quantitative PCR approach, this mimic is available on request from the author. 4. Reverse transcription with a random hexamer will target all RNA species. Specific primers for the product of interest, out with the PCR product sequence, or polyA primers to target mRNA may be substituted as required. 5. Because of the high activity of 32 P, alternatives such as 33 P or biotin-dCTP may be considered. 6. Magnesium chloride concentrations may need to be altered for different PCR products. We therefore recommend using buffer with separate magnesium chloride. 7. Using heated lids avoids the need for paraffin oil overlay, which reduces evaporation. 8. To evaluate the reproducibility of the reverse transcription reaction we recommend including 0.5 µCi [ 35 S]dATP. This small amount of [ 35 S]dATP does not affect subsequent quantitation of the PCR. In our previous experiments, we demonstrated an intra-assay variation of 17% between samples in RT reactions and a 9% mean inter assay variation. 9. The sensitivity of the assay can be varied by varying the standard range; decreasing the range of mutant and control plasmid from 0.1 to 100 pg to 0.01 pg to 1.0 pg increased the sensitivity of detection. For these experiments, PCR was performed over 30 cycles. 10. The restriction enzyme EcoRV has an optimal digestion in buffered 50 mM KCl, 10 mM NaCl, and addition of 10% 100 mM NaCl to the PCR mix best approximates these conditions and avoids the need for purification of the PCR product at this step. The use of amplified control and mutant plasmids provides a high degree of control over the efficiency of the restriction digestion reaction.
210 Bartlett 11. Cerenkov counting is a simple principle where excised gel fragments are counted in the absence of scintillation fluid. Image analysis of the exposed autoradiograph can also be used at this stage. 12. The assay has not been evaluated below this limit and theoretically could function at sensitivities of tens or hundreds of mRNA copies; at these low concentrations, stochastic errors may be introduced. 13. Where all samples from a given individual or experiment are included in the same assay, interassay variation can be ignored. References 1. Bartlett, J. M. S., Hulme, M. J., and Miller, W. R. (1996) Analysis of cAMP RI-alpha messenger-RNA expression in breast cancer—Evaluation of quantitative polymerase chain-reaction for routine use. Br. J. Cancer 73, 1538–1544. 2. Bartlett, J. M., Wu, F. C. W., and Sharpe, R. M. (1987) Enhancement of Leydig cell testosterone secretion by isolated seminiferous tubules during co-perifusion in vitro: comparison with static co-culture systems. Intl. J. Androl. 10, 603–617. 3. Bartlett, J. M. S., Weinbauer, G. F., and Nieschlag, E. (1989) Quantitative analysis of germ cell numbers and relation to intratesticular testosterone following vitamin a-induced synchronization of spermatogenesis in the rat. J. Endocrinol. 123, 403– 412. 4. Touraine, P., Martini, J. F., Zafrani, B., Durand, J. C., Labaille, F., Malet, C., et al. (1998) Increased expression of prolactin receptor gene assessed by quantitative polymerase chain reaction in human breast tumors versus normal breast tissues. J. Clin. Endocrinol. Metab. 83, 667–674. 5. Sugimoto, T., Tsukamato, F., Fujita, M., and Takai, S. (1994) Ki-ras and c-myc oncogene expression measured by coamplification polymerase chain reaction. Biochem. Biophys. Res. Commun. 201, 574–580. 6. Bieche, I., Olivi, M., Champeme, M. H., Vidaud, D., Lidereau, R., and Vidaud, M. (1998) Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer. Intl. J. Cancer 78, 661–666. 7. Sandberg, M., Skalhegg, B., and Jahnsen, T. (1990) The two mRNA forms for the type I alpha regulatory subunit of cAMP-dependent protein kinase from human testis are due to the use of different polyadenylation site signals. Biochem. Biophys. Res. Commun. 167, 323–330.
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63. Primed In Situ Nucleic Acid Lab
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18 McDonagh stored. This means that
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28 Bartlett References 1. US Depart
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34 Pearson and Stirling 11. Microfu
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36 Going 3. Methods 3.1. Section Cu
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42 Going
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44 Pearson 7. Add 60 µL of chlorof
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46 Bartlett 3. Methods 3.1. RNA Ext
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48 Pearson 3. Method The method of
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50 Stirling and Bartlett 4. Protein
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66 Bartlett Table 1 Recommended Aga
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68 Bartlett Table 2 Separation of D
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70 Bartlett 2. 5× TBE: 54 g of Tri
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74 Bartlett 4. Many modern electrop
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78 Stirling 11. Store at -20°C for
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82 Hyndman and Mitsuhashi 3.1. Effi
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84 Hyndman and Mitsuhashi Fig. 1. H
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102 Stirling Table 1 Thermal Cycler
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104 Stirling
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106 Wang and Young full-length cDNA
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108 Wang and Young Fig. 1. (A) Nort
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110 Wang and Young Fig. 3. Expresse
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112 Wang and Young 2. First-strand
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116 Wang and Young
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118 Dassanayake and Samaranayake co
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120 Dassanayake and Samaranayake 5.
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124 Iannone et al. Fig. 1. Diagram
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Table 1 Oligonucleotides Descriptio
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128 Iannone et al. 5. For microsphe
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130 Iannone et al. 4. To adjust for
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132 Iannone et al. 6. Target concen
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152 Olmos et al. Fig. 1. RT-hemines
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154 Olmos et al. This nested RT-PCR
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Page 161 and 162: 162 Abe 5. Moloney murine leukemia
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278 Oien 50-mL conical tubes. Resus
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282 Oien 8. PCR. With SAGE, achievi
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288 Edwards and Bartlett technology
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386 Walsh by most, but not all M13
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392 Walsh
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452 Gilchrist and Befus References
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474 Wang
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486 Preston amplification approach
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492 Preston 2. Remove the AmpliTaq
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494 Preston 3.3. Cloning and DNA Se
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498 Preston 21. Hung, T., Mak, K.,
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504 Ravassard et al. 9. ddH 2 O. 10
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506 Ravassard et al. 3.2.2. RNA Mix
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508 Ravassard et al. 4. Wash twice
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510 Ravassard et al.
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512 Pont-Kingdon Fig. 1. Constructi
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530 Burke and Barik purified mutant
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532 Burke and Barik
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