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Technical Briefs |
1
Institute of Clinical Chemistry and Laboratory Medicine, Westfälische Wilhelms Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Muenster, Germany;
2
European Laboratory Association, Section Ibbenbueren, D-49477 Ibbenbueren, Germany;
3
Department of Oral and Maxillofacial Surgery, Westfälische Wilhelms Universität Münster, D-48149 Muenster, Germany;
4
Institute of Immunology, University of Witten/Herdecke, D-58453 Witten, Germany;
a author for
correspondence: fax 49-251-83-4-7226, e-mail brandt{at}uni-muenster.de
c-myc gene amplification that leads to overexpression has been shown to play a major role in cancer development, especially in breast cancer (1). Two methodological approaches based on PCR, differential PCR (2) and competitive PCR (3), have been developed for gene dosage quantification and have been applied to c-myc. When these methods are used, both degraded DNA and the necessarily low competitor concentrations, which are prone to dilution errors, lead to over- or underestimation of gene dosages. Methods using separate comparisons of two single-copy reference genes for any DNA sample (double-differential PCR) (4)(5), dilution series of the competitors (competitive-differential PCR) (6), or sample DNA (in differential PCR) (7) have been introduced to quantitative PCR to circumvent those problems. In consequence, the methods are labor-intensive, time-consuming, and expensive.
We improved quantitative PCR and developed a reliable and sensitive direct-double-differential PCR method for c-myc gene dosage quantification in which DNA fragments of two different single-copy reference genes, manganese superoxide dismutase (SOD2) and ß-globin (HBB), and the target DNA fragment (c-myc) are coamplified simultaneously in one reaction tube. The c-myc PCR product (110 bp) is bracketed by the SOD2 (90 bp) and HBB (252 bp) PCR fragments in length. Sequences were exponentially amplified using 40 ng of sample DNA in a 50-µL reaction volume under the following reaction conditions: denaturation at 94 °C for 3 min; followed by 25 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 62 °C, and extension for 1 min 72 °C; and a final extension step of 7 min at 72 °C.
To exclude incorrect gene dosage quantification caused by varying PCR
reactant concentrations (8), all PCR components [10 pmol of
primer (shown in Table 1
); a downstream primer labeled fluorescently with
6-carboxy-fluorescein (FAM; Biometra); 0.3 µL of Taq DNA Polymerase
and 1x Taq reaction buffer with MgCl2 (Pharmacia);
and 200 µmol each of GeneAmp® dNTPs (Perkin-Elmer)]
were premixed and applied to equal amounts of patient DNA, leukocyte
DNA, and a high-copy DNA sample from SK-BR-3 cells. Formalin fixation,
paraffin embedding, isolation, and long-term storage of DNA are
associated with DNA fragmentation. In addition, PCR product yield is
inversely correlated to amplicon length, especially in degraded DNA
(9). Therefore, the peak area of a single-copy
c-myc gene dosage was calculated by using the length and
peak areas obtained from GenescanTM 2.1 evaluation
software (Perkin-Elmer) of the two control fragments (Fig. 1
, A and B) as two points on a line. This line allowed the
determination of the peak area of the single-copy gene (Ac-myc
single-copy gene) at the length of the
c-myc amplicon (Fig. 1C
) as described in Eq. 1 .
 | (1) |
|
|
where LSOD2 is the length of SOD2 in base pairs; LHBB is the length of HBB in base pairs; Lc-myc is the length of c-myc in base pairs; AHBB is the peak area of HBB; and ASOD2 is the peak area of SOD2.
The gene dosage was obtained by dividing the detected peak area
(Ac-myc det) by the calculated peak area
(Ac-myc single-copy gene) of the
c-myc PCR product (Eq. 2 ).
 | (2) |
The accuracy of our method was tested in 10 independent analyses
of leukocyte DNA, which revealed an average gene dosage of 1.12 (n
= 10; range, 1.03–1.16; CV = 4%). When we used degraded
leukocyte DNA (as documented by agarose gel electrophoresis), our
method exhibited a gene dosage for the c-myc gene of 1.08
(n = 3; range, 0.97–1.14; CV = 7%), which facilitates the
gene dosage quantification of highly degraded DNA such as DNA in plasma
or paraffin-embedded tissue. Examples of single-copy and increased
c-myc gene dosages are shown in Fig. 1
, A and B. When we
applied this method to the analysis of c-myc gene dosages of
breast cancer patients as well as to the breast cancer cell line
SK-BR-3, we could detect a two- to fourfold increase of the
c-myc gene copy number in 3 of 20 cases and a 10-fold
increase of the c-myc gene copy number in SK-BR-3 (n =
3; range, 9.31–10.26; CV = 4%), which is in agreement with data
in the literature (1)(10).
This method, which uses two control amplicons in combination with standardized laser-induced capillary electrophoresis, allows fast, reliable, and sensitive quantification of gene dosages without the use of synthetic competitors, even in the presence of highly degrade DNA.
Acknowledgments
We thank Gerd Assmann (Institute of Clinical Chemistry and Laboratory Medicine, Westfälische Wilhelms Universität, Münster) for financial support.
References
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double-differential-PCR for
c-myc.