Detection of Arg72Pro Polymorphism of the Tumor Suppressor Gene (TP53) by a Rapid One-step Tetra-primer Amplification Refractory Mutation System-PCR

MATERIALS AND METHODS

Subjects: The population studied consisted of 92 white healthy unrelated subjects living in Aleppo, Syria (50 males, 42 females, Mean age 41±10). The participants were not previously diagnosed with cancer. All subjects were native Syrians. Informed consent was obtained from each subject. Blood samples were collected in EDTA tubes and anonymously coded and stored.

DNA isolation: Genomic DNA was isolated from 200 μL venous blood using a spin column format kit (EuroGold™ Blood DNA mini kit, Euroclone, Italy). The procedure was carried out according to the manufacturer recommendations.

Quantification of Genomic DNA: Quantification of DNA was performed using V-650 UV-VIS spectrophotometer (Jasco, Japan). A260/A280 ratios were in the range of 1.8-1.9 indicating high purity Genomic DNA extract. Extraction yield was in the range of 4-8 μg per 200 μL blood.

Tetra primer ARMS-PCR analysis: Four primers were used in a single PCR reaction (schematic illustration in Fig. 1). Two primers, P1 and P2 (Table 1), were designed to amplify a 281 bp band which served as a control for the success of amplification. Two specific primers, P3 and P4, with complementary 3'-terminal nucleotide to the corresponding polymorphism, were introduced (Table 1). To enhance the specificity, a destabilizing mismatch was incorporated at the 3rd nucleotide from the 3'-terminus in each primer.

The PCR reaction was carried out in a total volume of 30 μL, containing 50-100 ng of genomic DNA as template, 0.125 μM of P1 and P2, 0.25 μM of P3 and P4, 0.2 mM of each dNTP, 2.4 mM of MgCl₂, Taq buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl) and 1.3 U of JumpStart^™ Taq DNA Polymerase (Sigma-Aldrich, USA).

Fig. 1:

Schematic illustration of the expected amplicons in ARMS analysis. Primers P2 and P1 always amplify a common region spanning the Arg72Pro pylomorphism. The amplification of a 193 or 131 bp band indicates the presence of either the Proline or Arginine allele, respectively

Fig. 2:

Agarose gel electrophoretogram of the TP53 Arg 72 Pro polymorphism analysis by tetra-primer ARMS-PCR for 8 different subjects. L: DNA ladder

Table 1:	Primers used in Arg72Pro TP53 analysis by tetra primer ARMS-PCR
Underlined bases indicate the introduced mismatches

PCR amplification was carried out in a MasterCycler^® thermal cycler (Eppendorf, Germany), with an initial denaturation at 94°C for 4 min, followed by 36 cycles of 94°C for 30 sec, 65°C for 30 sec and 72°C for 5 sec and a final extension at 72°C for 5 min. Six microliter of loading buffer was added to the PCR reaction and a 25 μL aliquot was subjected to agarose gel (2%) electrophoresis. The procedure rendered 3 bands in heterozygotes (281, 193 and 131 bp) and 2 bands in homozygotes (Arg/Arg resulting in 281 and 193 bp and Pro/Pro resulting in 281 and 131 bp) (Fig. 2).

Validation by PCR-RFLP analysis: Genotype assignment was validated for 8 samples using the PCR-RFLP method which was carried out as previously reported (Lee et al., 2000). The PCR product was digested with BstUI (Fermentas, Lithuania) according to the manufacturer’s directions. The digested PCR fragments were separated on 3.5% agarose gels. Genotypes were scored as homozygous for the Pro allele if the 199-bp PCR fragment after digestion remained uncut or homozygous for the Arg allele if after enzyme digestion two bands (113 and 86 bp) were observed. Gels with three DNA fragments (199, 113 and 86 bp) were scored as heterozygotes.

RESULTS AND DISCUSSION

Tetra primer-ARMS PCR analysis: PCR reaction conditions were thoroughly optimized to obtain the highest yield and specificity. The use of a HotStart method was found to be necessary in order to eliminate the formation of primer dimers increasing the yield of the informative amplicons. Figure 2 shows the electrophoretogram of tetra-primer ARMS-PCR products for 8 different genomic DNA samples. The three Arg/Arg, Arg/Pro and Pro/Pro genotypes were clearly distinguished. A common band of 281 bp size as the internal control appeared in all the cases and the presence of either the Arg or Pro variant rendered an additional fragment of 131 or 193 bp, respectively. Analysis by PCR-RFLP proved the validity of the proposed method.

RFLP-PCR has been the traditional method for the detection of Arg72Pro (Lee et al., 2000; Belyavskaya et al., 2006; Costa et al., 2008). The several steps associated with the use of this method, especially restriction enzyme digestion, increase analysis time. By contrast, the proposed method involves a single PCR step. The PCR-SSCP method (Noma et al., 2004) is difficult to implement and may require re-optimization. By contrast, the proposed method is simple, robust and demands low level of expertise.

The speed, simplicity, ease of use, cost-effectiveness and high accuracy of the proposed method make it suitable for the analysis of very large number of samples, which is a demand for reliable case-control and population genetic studies.

Genotype distribution of Arg72Pro polymorphism in a sample population from syria: A total of 92 subjects living in Aleppo were genotyped for the Arg72Pro polymorphism, which is known to be differentially distributed among different populations. Among the 92 subjects genotyped, 47 (51%) were homozygous for the Arg allele, 11 (12%) were found to be homozygous for the Pro allele and 34 (37%) were found to be heterozygous. The genotype distribution was found to be in Hardy-Weinberg equilibrium (χ² = 1.49; df = 2; p = 0.47) (Table 2).

Table 2:	Genotype and allele distribution for Arg72Pro TP53 polymorphism in a syrian population

ACKNOWLEDGMENT

We are thankful to Dr. A-E. Al Moustafa for his critical reading of the manuscript.