Rapid Direct PCR for ABO Blood Typing*

Abstract

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

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

“…DNA was extracted using a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. ABO genotypes of all DNA samples were determined using the allele-specific PCR method [20]. The ABO genotypes of these 80 samples included 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).…”

Evolution of technology for molecular genotyping in blood group systems

“…ABO genotyping using fresh blood, hair, body fluids, etc . without extracting DNA has also been described 41 where, a fast PCR instrument and optimized Taq polymerase were used. The amplified products were analysed by GeneScan programme after capillary electrophoresis.…”

Genetic Kinship Investigation from Blood Groups to DNA Markers

“…Forensic use of the ABO system was finally simplified and accelerated thanks to more advanced PCR techniques. For example, in 2011, Lee et al [19] announced a rapid direct ABO genotyping method that eliminates the need for DNA extraction, using a multiplex allele-specific primer set for the simultaneous detection of 3 single nucleotide polymorphism (SNP) sites. In 2011, another group, Watanabe et al [20], developed a simple method for ABO genotyping using a DNA chip.…”