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Technical Briefs |
G Mutation of Medium-Chain Acyl-CoA Dehydrogenase Gene by Real-Time PCR
1
Servicio de Bioquímica, Hospital Universitario Virgen del Rocío, Avda/Manuel Siurot s/n, 41013 Seville, Spain
2
Biomedal, CE Pabellón de Italia, C/Isaac Newton s/n, 41092 Seville, Spain
aauthor for correspondence: fax 34-954081279, e-mail liban{at}inicia.es
Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the
most common hereditary defect of fatty acid oxidation in humans. This
deficiency is an autosomal recessive disorder clinically characterized
by episodic hypoglycemia, encephalopathy, apnea, and sudden death among
children (1). A single A-to-G nucleotide transition at
position 985 (nt c985 A
G) of the MCAD gene represents
>81% of alleles causing MCAD deficiency (2). The frequency
of this allele variant exhibits considerable geographical variation
with a high prevalence in Northern Europeans (3).
PCR-based technologies are now widely used for the identification of
the nt c985 AG mutation for the MCAD deficiency
(4)(5); however, they involve multiple steps and
are time-consuming.
We used real-time PCR amplification coupled to fluorescence resonance
energy transfer and melting curve analysis (6) to detect nt
c985 AG mutation of the MCAD gene using the single-step
LightCycler technology.
In this study, we used genomic DNA isolated from EDTA blood from individuals who had been typed previously by PCR-restriction fragment length polymorphism analysis as was described by Matsubara et al. (7). DNA was isolated using the High Pure PCR Template Preparation reagent set (Roche Diagnostics) according to the manufacturer’s instructions. PCR was performed in a reaction volume of 10 µL with 0.5 µM each of the primers 5'-AGCACCAAGCAATATCATTTATG-3' and 5'-GCCTCCAAGTATCTGCACAG-3', 50 ng of genomic DNA, and 0.5 µM each of the anchor and detection probes (TIB MOLBIOL). The anchor probe 5'-CCAAGCTGCTCTCTGGTAACTCATT-3' was labeled at the 3' end with fluorescein; the sensor probe 5'-AGCTAGTTCAACTTTCATTGCCAT-3' was labeled with LightCycler Red 640 at its 5' end and modified at its 3' end by phosphorylation to block extension. As reaction buffer in the PCR, the LightCycler DNA-Master hybridization Probes 10x buffer (Roche Diagnostics) with a final MgCl2 concentration of 3.5 mM was used. Cycling conditions were as follows: 95 °C for 1 min; and 40 cycles of 95 °C for 0 s, 59 °C for 20 s, and 72 °C for 20 s (ramping rate, 20 °C/s). Fluorescence was monitored at the end of each 20-s annealing phase. After amplification, melting curves were generated by denaturation at 95 °C for 0 s, holding the samples at 50 °C for 20 s, and then heating the sample to 75 °C at 0.2 °C/s, simultaneously monitoring the decline in fluorescence. Melting curves were converted to melting peaks by calculating the negative derivative of the fluorescence with respect to temperature (-dF/dT) against temperature (T).
Typical results for genotyping using this method are shown in Fig. 1
. The melting peak of the wild-type sample (curve 1) was at
66.9 °C, whereas the mutant homozygous sample (curve 2) produced a
melting peak at 64.5 °C. The heterozygous sample produced two
melting peaks at 66.9 and 64.5 °C (curve 3).
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The whole process, including DNA extraction, was completed within 70 min.
With this method we analyzed 18 individuals from four different MCAD-deficient families and 25 healthy controls (50 chromosomes). The results were consistent with those obtained previously by restriction fragment length polymorphism analysis.
In conclusion, this new method combines simple sample processing and rapid analysis; it therefore affords both high-throughput genotyping and rapid results.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Gonzalez, M.E. Saez, M.J. Aragon, J.J. Galan, P. Vettori, L. Molina, C. Rubio, L.M. Real, A. Ruiz, and R. Ramirez-Lorca Specific haplotypes of the CALPAIN-5 gene are associated with polycystic ovary syndrome Hum. Reprod., April 1, 2006; 21(4): 943 - 951. [Abstract] [Full Text] [PDF]
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 A. Gonzalez, E. Abril, A. Roca, M. J. Aragon, M. J. Figueroa, P. Velarde, J. L. Royo, L. M. Real, and A. Ruiz CAPN10 Alleles Are Associated with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3971 - 3976. [Abstract] [Full Text] [PDF]
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