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Articles |
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Servizio di Genetica Medica, IRCCS Burlo Garofolo, 34137 Trieste, Italy
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Sezione di Genetica Medica, Dipartimento Scienze della Riproduzione e dello Sviluppo, Università di Trieste, 34137 Trieste, Italy
a address correspondence to this author at: Servizio di Genetica, IRCCS Burlo-Garofolo, Via dell’Istria 65/1, 34137 Trieste, Italy; fax 39-040-3785210, e-mail crovella{at}burlo.trieste.it
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Introduction |
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 Top Introduction References |
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The AGT enzyme is encoded by a single-copy gene (AGXT), which consists of 11 exons ranging from 65 to 407 bp and spanning a 10-kb DNA segment in the 2q37.3 human region. AGT is a 392-amino acid protein with a molecular mass of 43 kDa (2).
Several technical approaches have been used to identify 7 polymorphisms and 26 mutations in the AGXT gene (3)(4)(5)(6). Here we describe a rapid, flexible, and inexpensive method for detection of the different types of mutations (insertions, deletions, point mutations) of the AGXT gene. Our method is based on the ability to distinguish between PCR amplification products by their melting temperatures (Tm) (7)(8)(9).
Nine PH1 patients, whose mutations had first been analyzed by the single-strand conformation polymorphism (SSCP) technique and then by sequencing of abnormal mobility bands of four AGXT exons (5), were studied comparatively by the melting temperature assay (MTA). Heterozygous relatives of three patients were also included in this study. Five healthy Italian subjects served as wild-type controls. The clinical diagnosis of PH1 was based on previously described criteria (5).
DNA was isolated from EDTA-collected peripheral whole blood by standard laboratory techniques. Amplification of the four exons (exons I, II, IV, and X) of the AGXT gene, which represent the majority of the Italian mutations, was performed as described previously by Pirulli et al. (5). For the melting detection assay, the PCR reaction was optimized according to the method described by Marziliano et al. (9).
After optimization, PCR was performed in a final volume of 50 µL with 1x SYBR Green I Buffer (PE Biosystems); 5.5 mmol/L MgCl2; 200 µmol/L each of dATP, dGTP, and dCTP; 400 µmol/L dUTP; 5 U of AmpliTaq Gold (PE Biosystems); 1 U of AmpErase UNG (PE Biosystems); and 50 ng of genomic DNA. PCR was performed using the following primers:
The following cycling conditions were used: 50 °C for 2 min, 95 °C for 10 min, 94 °C for 15 s, and 60 °C for 1 min, repeated 40 times. As indicated by Marziliano et al. (9), the dissociation protocol included a slow cooling from 95 °C to 60 °C over a period of 20 min (during this time, a fluorescent point was detected every 3 s for a total of 400 data points) at the end of PCR. The reaction was performed on the ABI 7700 Sequence Detection System (PE Biosystems) equipped with the 7700 Sequence Detection System Software, Ver. 1.7. At the end of amplification (40th cycle), the melting profile of each amplicon was analyzed with this software, which allowed us to identify the point at which the reassociation occurred (flexus point). Samples were analyzed in quadruplicate and in different PCR runs to test the reproducibility of this technique.
We used 50 ng of genomic DNA for MTA genotyping. An optimized PCR is crucial for the correct evaluation of the MTA profile of the amplicons. For this reason, we ran a primer optimization matrix in quadruplicate, using final concentrations of 50 and 300 mmol/L for each primer and the ABI 7700 Sequence Detection System in "real-time" data collection.
To verify formation of nonspecific amplicons, no-template controls were run together on the same assay. The 300/50 mmol/L, 50/300 mmol/L, and 50/50 mmol/L forward/reverse primer combinations did not reveal any nonspecific amplicons. At the end of 40 PCR cycles with no-template controls, no fluorescence signal was detected. When the DNA target was present, the maximum efficiency of PCR with no background noise attributable to primer dimers or nonspecific amplicons was reached after 36 cycles for exon I, 34 cycles for exon II, 35 cycles for exon IV, and 30 cycles for exon X. The optimal primer combinations 50/300 mmol/L (exons I and II) and 300/50 mmol/L (exons IV and X) were chosen because lower cycle thresholds indicate a higher sensitivity of the test (9).
As reported previously by Heid et al. (10) and Marziliano et al. (9), it possible to establish the right amount of starting genomic DNA by analyzing the cycle-by-cycle PCR product formation.
During the dissociation protocol (slow cooling from 95 °C to 60 °C over a period of 20 min), the ABI 7700 instrument was also used in real time (every 3 s) to follow the melting profile of each sample. The correct melting peak for each sample was calculated by an appropriate algorithm as the first derivative at the flexus point (11).
The flexibility of MTA allowed us to easily distinguish different types
of AGXT mutations, including single-base substitution
(G243A, G468A, T576A, G630A), single-base deletions (C155del,
G1098del), a single-base insertion (C156ins), a 3-base insertion
(GAG408ins), and double substitutions (G630A-G640A, G244C-C252T) in
both heterozygotes or homozygotes (Table 1
).
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MTA technology allowed us to detect mutations G468A, C155del, and C156ins, which had not been detected by SSCP analysis. In particular, the presence of an extra C in position 156 caused an increase of the Tm (88.0 ± 0.13 °C and 88.8 ± 0.2 °C, respectively, for heterozygous and homozygous conditions, compared with 87.3 ± 0.14 °C for the wild type). Similarly, the deletion of C in the same region created a decrease of Tm (86.8 ± 0.11 °C and 86.1 ± 0.09 °C, respectively, for heterozygous and homozygous conditions, compared with 87.3 ± 0.14 °C for the wild type). Moreover, the G468A mutation in the heterozygous condition reduced the Tm to 87.9 ± 0.01 °C from 88.4 ± 0.1 °C for the wild type. The other AGXT mutations highlighted by MTA were also detected by SSCP analysis. The five healthy controls showed the same Tm values in the four exons studied, indicating the reproducibility of this test.
In this study, we evaluated the sensitivity, specificity, and possibility of automating MTA for the detection of different types of AGXT mutations. To date, the molecular diagnosis of PH1 has been based mainly on the screening of PCR products by polyacrylamide gel electrophoresis. This approach is labor-intensive and time-consuming. MTA technology for AGXT mutation detection thus might become a powerful and sensitive tool for rapid screening. In MTA, the amplified product (labeled with SYBR Green I) is directly and automatically analyzed by the ABI 7700, whereas SSCP is less sensitive, more time-consuming, and requires highly purified PCR products that must be run on polyacrylamide gels.
MTA analysis can be easily automated, and a large number of samples can
thus be processed without operator input (e.g., a 96-well plate is
analyzed in 
2 h). The melting profiles are generated by appropriate
software without use of operator time.
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Acknowledgments |
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References |
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TopIntroductionReferences |
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