A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes.
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 3h. 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.
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea.
Kyungmyung
K
Lee
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea.
Hyun-Chul
HC
Park
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea.
Sanghyun
S
An
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea.
Eu-Ree
ER
Ahn
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea.
Yang-Han
YH
Lee
DNA Analysis Section, Busan Institute of National Forensic Service, 50 Kumoh-ro, Mulgumeup, Yangsan-si, Gyeongsangnam-do 626-742, South Korea.
Mi-Jung
MJ
Kim
DNA Analysis Section, Gwangju Institute of National Forensic Service, 111 Daedeok-ri, Seosam-myeon, Jangseong-gun, Jeollanam-do 515-822, South Korea.
Eun-Jung
EJ
Lee
SeaSun Biomaterials, N517 Daedoek Campus, Pai Chai University, 11-3 Tekeuno 1-ro, Gwanpyeong-dong, Yuseong-gu, Daejeon 305-509, South Korea.
Jae Sin
JS
Park
SeaSun Biomaterials, N517 Daedoek Campus, Pai Chai University, 11-3 Tekeuno 1-ro, Gwanpyeong-dong, Yuseong-gu, Daejeon 305-509, South Korea.
Jin Wook
JW
Jung
Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do 220-170, South Korea. Electronic address: neobios@korea.kr.
Sikeun
S
Lim
D000017:ABO Blood-Group System / Q000235:genetics*; D003415:Crime; D005453:Fluorescence; D005554:Forensic Medicine / Q000379:methods; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D006824:Hybridization, Genetic; D020135:Peptide Nucleic Acids / Q000032:analysis*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction
D000017:ABO Blood-Group System; D020135:Peptide Nucleic Acids
ABO genotyping; Fluorescence melting curve analysis; Peptide nucleic acid; Real-time polymerase chain reaction; Single nucleotide polymorphism