A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

Highlights

• We developed PNA probe-based FMCA for ABO genotyping.

• This method allowed for simultaneous detection of three SNP sites in the ABO gene.

• Eighty samples were successfully genotyped.

• Reactions were performed in one single closed tube in less than 3 h.

• This method may be an effective and helpful tool in forensic investigations.

Abstract

ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n = 5], A/O01 or A/O02 [n = 23], B/B [n = 3], B/O01 or B/O02 [n = 18], A/B [n = 9], O01/O01 or O01/O02 or O02/O02 [n = 20], cis-AB01/A [n = 1], and cis-AB01/O01 or cis-AB01/O02 [n = 1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3 h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.

Introduction

ABO typing is commonly used in forensic investigations, including the identification of decomposed bodies and the screening of suspects in crime investigations. ABO typing is generally performed using serological methods, which limit the use of minute, degraded, contaminated, and poor-quality samples in forensic casework. However, ABO genotyping of DNA can be applied to most forensic casework samples, including bones, decomposed organs, and semen-contaminated vaginal fluid.

Since Yamamoto and colleagues reported the differences in the nucleotide sequences of ABO alleles [1], [2], numerous methods for ABO genotyping based on polymerase chain reaction (PCR) have been developed, such as PCR-restriction fragment length polymorphism (PCR-RFLP) analysis [3], mutagenically separated PCR (MS-PCR) [4], multiplexed allele-specific PCR [5], [6], PCR with sequence-specific primers (PCR-SSP) [7], [8], universal reporter primer systems [9], and real-time PCR using allele-specific primers [10]. However, most of these methods require multiple post-PCR manipulation steps, including electrophoresis and enzyme digestion; for this reason, these methods often have a risk of carry-over contamination [10].

Recently, probe-based fluorescence melting curve analysis (FMCA) has been reported as a powerful tool for the detection of single nucleotide polymorphisms (SNPs); this detection is based on melting temperature (T_m) shifts due to thermal denaturation of the probe-target hybrid [11]. FMCA provides closed-tube SNP genotyping without post-PCR manipulations. Therefore, FMCA reduces analysis time and the risk of carry-over contamination [12], [13].

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which a negatively charged sugar-phosphate backbone has been replaced by a synthetic peptide backbone, which is usually composed of repeating N-(2-aminoethyl)-glycine units [14], [15], [16]. The uncharged and flexible synthetic backbone provides remarkably high affinity and specificity when PNAs hybridize to complementary target nucleic acid sequences. Therefore, PNA–DNA duplexes are much more stable than the corresponding DNA–DNA duplexes. Furthermore, since PNA–DNA hybridizations in single-base mismatches are quite unstable, PNAs can be used as powerful molecular hybridization probes to detect SNPs and point mutations. Thus, several PNA-dependent methods have been developed in molecular genetics and cytogenetics [17], [18], [19].

In this study, we describe a new method for ABO genotyping using PNA probe-based FMCA. This method allowed simultaneous detection of three SNPs (nucleotide positions 261, 526, and 803) and identified A, B, O01 or O02, O03, and cis-AB01 alleles in the ABO gene.