Duplex Detection of TP53 Arg72Pro and 16 bp Del/Ins Polymorphisms by a Simple Optimized PCR-RFLP Method.

BACKGROUND: The tumor suppressor gene (TP53) encodes p53, the central protein in the apoptotic pathway which has been shown to be of crucial importance in the development of cancers in addition to a variety of neurodegenerative disorders. Two most commonly studied polymorphisms that were shown to affect the biochemical functions of p53 protein are the exon 4 Arg72pro and Intron 3 16 bp Del/Ins polymorphisms. AIMS: The aim of the present work is to develop a new optimized method for the simultaneous detection of the two important polymorphisms in the TP53 gene in a single reaction.
MATERIALS AND METHODS: The proposed method is based on amplification of a single PCR amplicon and the use of a unique restriction enzyme with restriction sites that facilitate simultaneous detection.
RESULTS: The proposed method offers fast, economical, and simple simultaneous detection. Validation by methods commonly used in the literature showed perferct concordance in genotyping results.
CONCLUSION: The proposed method can serve as an invaluable tool for the investigation of TP53 Arg72Pro-16 bp Del/Ins haplotype, and the combined effects of the two polymorphisms offering extreme ease and simplicity over the currently used methods which are based on two separate detections.

Introduction

The human TP53 tumor suppressor gene is located on chromosome 17p13 and encodes a 53 kDa nuclear phosphoprotein, which plays a central role in many cellular processes, such as cell-cycle control, DNA repair, and apoptosis.[1] Since TP53 plays an important role in cell cycle regulation and in maintenance of genome stability by preventing mutations, it is often referred to as the “guardian of the genome.n”[2] Loss or mutation of TP53 is probably the commonest single genetic change in cancer.[3-7]

Several polymorphisms have been detected within the TP53 gene. A common TP53 polymorphism at codon 72 of exon 4 (dbSNP ID: rs1042522), designated as Arg72Pro, has been reported to modify the risk and/or prognosis of many types of cancer.[8-12] This polymorphism derives from a single-nucleotide substitution at codon 72, where either CCC encodes proline or CGC encodes arginine, resulting in a non-conservative change. This polymorphism is located in a proline-rich domain of p53, which is known to be important for the growth suppression and apoptotic functions.[13] Evidence is emerging indicating that the Arg allele and the Pro allele of the TP53 codon 72 polymorphism are not equivalent in biochemical property and function.[14]

The most commonly investigated intronic polymorphism in the TP53 gene is intron 3 16 bp duplication (dbSNP ID: rs17878362), designated as IVS3 16 bp Del/Ins. Mutations and polymorphisms in intron sequences of the TP53 gene may affect pre-messenger RNA (mRNA) splicing, which may affect mRNA translation.[15] Furthermore, intronic polymorphisms may influence coding-region mutations that increase the likelihood of a deleterious phenotype.[16] Because introns have also been implicated in regulating gene expression and DNA–protein interactions,[17-19] mutations and polymorphisms in intron sequences may affect these functions. Indeed, the IVS3 16 bp Del/Ins polymorphism of the TP53 gene was found to be associated with several types of cancer.[20-23]

Several methods for the separate detection of the two polymorphisms have been described. The most commonly used method for the detection of Arg72Pro is based on the principle of PCR-RFLP.[2425] Tetra primer AMRS-PCR have also been utilized for the detection of Arg72Pro.[26] Intro 3 16 bp Del/Ins polymorphism has been detected based on amplifying the region containing the Del/Ins polymorphism by a separate PCR and observing the length of the PCR product by gel electrophoresis.[27] To our knowledge, no previous method for the simultaneous detection of the two polymorphisms in a single PCR reaction has been described. The aim of the present work is to develop a simple RFLP-PCR method for the simultaneous detection of the two polymorphisms, which are very frequently investigated in combination, in order to cut analysis time and cost in half.

Materials and Methods

DNA Extraction

DNA was extracted from 200 μl aliquots venous blood using QIAamp™ Blood Mini Kit (Qiagen, Germany) according to the manufacturer's recommendations. The quantity and quality of the extracted DNA were checked using UV absorption measurement at 260 nm (Jasco spectrophotometer V-600, Japan) and agarose gel electrophoresis, respectively.

Duplex PCR-RFLP Detection

Figure 1 shows a schematic illustration of the theoretical basis of the simultaneous detection of TP53 Arg72Pro and intron 3 16 bp Del/Ins polymorphisms by the proposed method. The presence of multiple restriction sites for the BsaJI restriction enzyme in strategic positions with respect to the two polymorphisms was utilized to detect the Arg72Pro polymorphism and facilitate the detection of the intron 3 16 bp Del/Ins polymorphism following the amplification of a single PCR product containing both polymorphisms.

Figure 1

A schematic illustration showing the basis of the simultaneous detection of TP53 Arg72Pro and Intron 3 16 bp Del/Ins polymorphisms by a single restriction enzyme. Filled triangles represent constant restriction sites. The half-filled triangle represents the TP53 Arg72Pro polymorphic restriction sites. Primer positions are indicated by half arrows.

A single 315-bp PCR product was amplified using the forward primer: 5’-TCTGGTAAGGACAAGGGTTGG-3’ and the reverse primer: 5’-GGGAAGGGACAGAAGATGACAG-3’. PCR was carried out in a total reaction volume of 20 μl containing 0.5 μM of each primer, 50-150 ng of genomic DNA, 200 μM of each dNTP, 2.5 mM of MgCl2, 1X PCR buffer (10 mMTris-HCl, 50 mM KCl, 0.8% Nonidet P40) (Fermentas, USA), 1 unit of HotStart Plus Taq DNA polymerase (Qiagen, Germany), and 1 M of Betaine (SigmaeAldrich, USA). The PCR system TaKaRa PCR thermal cycler Dice (Takara BIO INC., Japan) was used for the amplification. PCR was commenced with a 5 min initial denaturation step at 95°C followed by 30 cycles of 94°C for 30 sec, 58°C for 30 s, and 72°C for 20 sec, and a final extension step at 72°C for 5 min. The resulting PCR product was digested with FastDigest® BsaJI (BseDI) (Fermentas, USA) by adding 1 μl of the restriction enzyme and 1.5 μl of 10× FastDigest® green buffer (supplied with the restriction enzyme) directly to the whole volume of the PCR reaction without purification. The mixture was then incubated at 37°C for 30 minutes. Alternatively, a standard BsaJI (BsedI) restriction enzyme can be used according to the manufacturer's instructions. The resulting fragments were separated by 3% agarose gel electrophoresis in 0.5× TBE buffer at 10 V/cm for 1 hour. Ethidium bromide was incorporated in the gel at a concentration of 1 μg/ ml. The gel was UV-illuminated and photographed using Alphalmager Mini system (ProteinSimple, USA).

Validation of the Proposed Method

The proposed method was validated for different samples by the commonly used methods performed as previously described.[2527] For the exon 4 codon 72 polymorphism, the primers (5’-CTGGTAAGGACAAGGGTTGG-3’ and 5’-ACTGACCGTGCAAGTCACAG-3’) amplified a 396 bp DNA fragment. The PCR product was digested with BstUI (Fermentas, USA). DNA from wild-type (G/G) homozygotes produced 165 bp and 231 bp bands; DNA from wild-type/mutant (G/C) heterozygotes produced 165 bp, 231 bp, and 396 bp bands; DNA from mutant (C/C) homozygotes produced a 396 bp band. For the intron 3 polymorphism, the primers (5’-TGGGACTGACTTTCTGCTCTT-3’ and 5’-TCAAATCATCCATTGCTTGG-3’) amplified 180 bp or 196 bp fragments. DNA from Del/Del homozygotes produced the 180 bp band; DNA from Del/Ins heterozygotes produced both bands; DNA from Ins/Ins homozygotes produced the 196 bp band.

Results

Following separation of the restriction fragments by agarose gel electrophoresis, similar band patterns to those expected based on Figure 1 were obtained. The presence of a 72Pro C-allele resulted into a 81 bp band. The presence of a 72Arg G-allele resulted into a 125 bp band. Heterozygousity for the Arg72Pro polymorphism resulted into both bands. Intron 3 16 bp Del/Ins polymorphism was detected simultaneously by the presence of a 140/156 bp fragment. Figure 2 shows an electrophoretogram for 8 different individuals showing all possible genotypes for the two polymorphisms.

Figure 2

Agarose gel electrophoretogram showing the simultaneous detection of TP53 Arg72Pro and intron 3 16 bp Del/Ins by the proposed PCR-RFLP method.

Genotype assignment was validated for 24 different samples using the commonly used methods. Validation showed 100% accordance in genotype assignment for both of the two polymorphisms.

Discussion

The currently used methods for the investigation of the combined effects of the two polymorphisms or their haplotype utilize two separate detections, one for each polymorphism.[24-27] The proposed method is the first to simultaneously detect the two most important polymorphisms of the TP53 gene which is a pivotal gene in apoptosis.

Through careful primer positioning and the use of a new restriction enzyme with unique restriction sites with respect to the region containing the two polymorphisms, it was possible to detect the two polymorphisms simultaneously. In particular, the presence of a constant restriction site for the chosen restriction enzyme in close proximity to the intron 3 16 bp Del/Ins polymorphism yields fragments with sufficient relative difference in length such that separation and detection of the 16 bp Del/Ins polymorphism by agarose gel electrophoresis is greatly facilitated.

Although PCR reaction conditions were optimized to ensure maximum efficiency and specificity, it should be emphasized that the use of a HotStart Taq DNA polymerase proved to be essential in order to reduce primer dimer formation and enhance amplification specificity. Furthermore, based on previous reports, 1M betaine was used to enhance digestion efficiency and increase PCR efficiency and specificity.[2829]

Our method can replace all the existing PCR-RFLP methods for the detection of Arg72Pro since it provides simple and simultaneous detection with the second most important polymorphism of the TP53 gene, the intron 3 16 bp Del/Ins polymorphism, and can be a valuable tool for the rapid detection of TP53 Arg72Pro – Intron 3 16 bp Del/Ins haplotype which is commonly studied in a wide variety of apoptosis-related medical conditions including cancer and neurodegenerative disorders.

Conclusion

A new method for the simultaneous detection of the two most commonly studied polymorphisms in the tumor suppressor gene (TP53) was described. The proposed method offers the advantage of simple and simultaneous detection of the two TP53 polymorphisms over the commonly used methods.