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366 Suomalainen and Syvänen
temperatures for the primer annealing may be required.
9. Streptavidin-coated microtiter plates made of scintillating polystyrene are available (Wallac,
Finland). In this case the final washing, denaturation and transfer of the eluted detection
primer can be omitted, but a scintillation counter for microtiter plates is needed (11).
10. The ratio between the cpm values for the two nucleotides reflects the ratio between the
two sequences in the original sample. Therefore, the solid-phase minisequencing method
can be used for quantitative PCR analyses (2–6). The R value is affected by the specific
activities of the [ 3 H] dNTPs used, and if either the mutant or the normal sequence allows
the detection step primer to be extended by more than one [ 3 H] dNTP, this will obviously
also affect the R value. Both of these factors can easily be corrected for when calculating
the ratio between the two sequences. Alternatively, a standard curve is constructed by
mixing the two sequences in known ratios and plotting the obtained R-values as a function
of the ratios to obtain a linear standard curve (3,5,6). The test results can then be interpreted
from the standard curve without the need of taking the specific activities of the number of
[ 3 H] dNTPs incorporated into account.
References
1. Syvänen, A.-C., Aalto-Setälä, K., Harju, L., Kontula, K., and Söderlund, H. (1990) A
primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E.
Genomics 8, 684–692.
2. Suomalainen, A. and Syvänen, A.-C. (2000) Quantitative analysis of human DNA sequences
by PCR and solid-phase minisequencing. Mol. Biotechnol. 15, 123–131.
3. Suomalainen, A., Kollmann, P., Octave, J.-N., Söderlund, H., and Syvänen, A.-C. (1993)
Quantification of mitochondrial DNA carrying the tRNA
Lys 8344 point mutation in myoclonus
epilepsy and ragged-red-fiber disease. Eur. J. Hum. Genet. 1, 88–95.
4. Syvänen, A.-C. (1999) From gels to chips: “Minisequencing” primer extension for analysis
of point mutations and single nucleotide polymorphisms. Hum. Mutat. 13, 1–10.
5. Syvänen, A-C., Söderlund, H., Laaksonen, E., Bengtström, M., Turunen, M., and Palotie, A.
(1992) N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase
minisequencing. Int. J. Cancer 50, 713–718.
6. Suomalainen, A., Majander, A., Pihko, H., Peltonen, L., and Syvänen, A.-C. (1993)
Quantification of tRNA
Leu 3243 point mutation of mitochondrial DNA in MELAS patients
and its effects on mitochondrial transcription. Hum. Mol. Genet. 2, 525–534.
7. Chen, X. and Kwok, P. Y. (1997) Template-directed dye-terminator incorporation (TDI)
assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy
transfer. Nucleic Acids Res. 15, 347–353.
8. Pastinen, T., Perola, M., Niini, P., Terwilliger, J., Salomaa, V., Vartiainen, E., et al. (1998)
Array-based multiplex analysis of candidate genes reveals two independent and additive
risk factors for myocardial infarction in the Finnish population. Hum. Mol. Genet. 7,
1453–1462.
9. Pastinen, T., Partanen, J., and Syvänen, A.-C. (1996) Multiplex, fluorescent solid-phase
minisequencing for efficient screening of DNA sequence variation. Clin. Chem. 42,
1391–1397.
10. Sitbon, G., Hurtig, M., Palotie, A., Lönngren, J., and Syvänen, A.-C. (1997) A colorimetric
mini-sequencing assay for the mutation in codon 506 of the coagulation factor V gene.
Thrombosis Haemostasis 77, 701–703.
11. Ihalainen, J., Siitari, H., Laine, S., Syvänen, A.-C., and Palotie, A. (1994) Towards automatic
detection of point mutations: use of scintillating microplates in solid-phase minisequencing.
BioTechniques 16, 938–943.