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Letters to the Editor |
1 Department of Medical Genetics, Free University of Brussels, Brussels, Belgium
2 Department of Human Genetics, CHU B35, University of Liège, Liège, Belgium
3 Department of Hemato-Oncology, University Hospital Erasme, Brussels, Belgium
aAddress correspondence to this author at: Department of Medical Genetics, Faculty of Medicine, Free University of Brussels, 808, route de Lennik, 1070 Brussels, Belgium. Fax 32-2-555-42-12; e-mail helhousn{at}ulb.ac.be.
To the Editor:
We developed a quantitative and very sensitive method that combines molecular beacon (1) and locked nucleic acid (LNA) technologies (2) in a single sealed tube. We used the oncogenic somatic JAK2V617F single-base sequence variant, observed in a broad range of Philadelphia chromosome-negative myeloproliferative diseases (MPDs) (3), to test this method of detection of somatic point variants. We used the molecular beacon to specifically detect the JAK2V617F mutant allele and the LNA oligonucleotide to limit amplification of the wild-type JAK2 sequences (see Fig. 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue7). With this method, we detected very small quantities of mutant alleles in tubes containing the homozygous JAK2V617F mutant cell line (HEL cell line) mixed with various amounts of wild-type cells and in clinical specimens containing small amounts of JAK2V617F mutated cells.
Primers and beacon probe were designed with the freeware MELT-CALC, Ver. 2.0 (http://www.meltcalc.de/). The melting temperature (Tm) of the molecular beacon stems was calculated by use of the Mfold algorithm (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi), whereas the Tm of the LNA was calculated via the Exiqon website (http://lna-tm.com).
We used unique PCR conditions (see Table 1 and experimental conditions in the online Data Supplement) that exploited the combined activities of the molecular beacon and LNA oligonucleotide to produce a fluorescent amplification signal highly specific to the JAK2V617F DNA target (Fig. 1A
). We tested different concentrations of LNA and found that a concentration of 1000 nM gave the most efficient blocking of the wild-type allele without significant alteration in the detection of the mutated allele (Fig. 1A
; also see Fig. 3 in the online Data Supplement). In addition, at this concentration, the low residual amount of wild-type allele present at the end of the PCR procedure was not recognized by the molecular beacon probe (Fig. 1A
).
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Dominguez and Kolodney (4) recommended the use of a mutated Taq polymerase, termed the Stoffel fragment, which lacks the 5'-3' exonuclease activity, on the basis of their hypothesis that this exonuclease activity would hydrolyze the LNA oligonucleotide and prevent it from blocking primer extension of the wild-type target template. We found, however, that the conventional Taq polymerase included in the commercially available Supermix-UDG (Invitrogen) did not significantly alter the blocking activity of the LNA component.
We tested the quantitative sensitivity of our method with serial dilutions of the HEL cell line DNA mixed with wild-type DNA, and the method was able to detect JAK2V617F mutated alleles at proportions as low as 0.01% (Fig. 1B
). The efficiency of this PCR was 91.9%, as demonstrated by the slope of the curve. Theoretically, the sensitivity could not be higher because, in a 100% homozygous mutant sample, a starting amount of 100 ng of genomic DNA would contain 
34 000 copies (based on a typical amount of 6 pg of DNA per diploid cell) of JAK2V617F mutant allele, and a diluted sample containing 0.01% of this mutant gene would theoretically represent 3 copies of the target sequence. We are thus very close to the limits of detection of any type of quantitative PCR assay. Of note, these results confirm that complete blocking of amplification of the wild-type allele is not an absolute prerequisite for very good quantitative sensitivity for the mutant allele.
We also compared the sensitivity of our tool with the Amplification Refractory Mutation System (ARMS)PCR method for JAK2V617F detection (5). We used ARMS-PCR to test DNAs from 23 patients carrying the JAK2V617F sequence variant and observed that all results were perfectly concordant with our method. Moreover, 2 patients with clearly positive results by our method showed only a weak signal by ARMS-PCR (data not shown). The great sensitivity as well as the quantitative aspects of our method present 2 advantages: (a) in some cases of positive JAK2V617F MPD, the variation is limited to small subclones; and (b) small molecules are being developed that specifically target the mutated protein (6). Our sensitive assay could be very useful in testing the efficacy of such molecules in the frame of therapeutic trials as well as in daily molecular follow-up.
In summary, we developed a very sensitive and rapid method that combines the advantages of LNA and molecular beacon technologies. Our tool, which can be used to detect any type of single-base variation, is a simple PCR performed in <40 min, under universal conditions and in sealed tubes, hence limiting the risk of false positivity and contributing to the high sensitivity offered by our PCR assay.
References
The following articles in journals at HighWire Press have cited this article:
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  K. Van Pelt, F. Nollet, D. Selleslag, L. Knoops, S. N. Constantinescu, A. Criel, and J. Billiet The JAK2V617F mutation can occur in a hematopoietic stem cell that exhibits no proliferative advantage: a case of human allogeneic transplantation Blood, August 1, 2008; 112(3): 921 - 922. [Full Text] [PDF]
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 T. S. Laughlin, M. W. Becker, J. L. Liesveld, D. A. Mulford, C. N. Abboud, P. Brown, and P. G. Rothberg Rapid Method for Detection of Mutations in the Nucleophosmin Gene in Acute Myeloid Leukemia J. Mol. Diagn., July 1, 2008; 10(4): 338 - 345. [Abstract] [Full Text] [PDF]
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E. Hammond, K. Shaw, B. Carnley, S. P'ng, I. James, and R. Herrmann Quantitative Determination of JAK2 V617F by TaqMan: An Absolute Measure of Averaged Copies per Cell That May Be Associated with the Different Types of Myeloproliferative Disorders J. Mol. Diagn., April 1, 2007; 9(2): 242 - 248. [Abstract] [Full Text] [PDF]
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), 10 nM (
), 100 nM (•), and 1000 nM LNA (
); wild-type DNA plus no (
) and 1000 nM LNA (
); and a control containing water and 1000 nM LNA (
). (B), amplification curves obtained from serial dilutions of homozygous JAK2V617F mutant allele diluted in wild-type DNA. As observed, a detection limit of 0.01% JAK2V617F could be reached. The dotted line indicates the fluorescent detection threshold. Shown are curves for 100% JAK2V617F (
); 0.5% JAK2V617F (
). Results of regression analysis: y = –3.538x + 31.255 (R2 = 0.9957).