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Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Published on: February 10, 2023

A simple ABO genotyping by PCR using sequence-specific primers with mismatched nucleotides.

Takashi Taki¹, Kazuhiko Kibayashi¹

¹Department of Legal Medicine, School of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.

Legal Medicine (Tokyo, Japan)

|March 25, 2014

Summary

Forensic ABO blood typing using PCR-SSP can be improved. Introducing mismatched nucleotides in primers reduces pseudo-positive signals, leading to more reliable ABO blood group determination from nail clippings.

Keywords:

ABO genotyping Allele-specific PCR Pseudo-positive signal

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Area of Science:

Forensic Science
Molecular Biology
Genetics

Background:

Accurate ABO blood group determination is crucial in forensic investigations.
Polymerase Chain Reaction with Sequence-Specific Primers (PCR-SSP) is a common method for ABO typing.
Pseudo-positive signals in PCR-SSP can compromise the accuracy of ABO typing.

Purpose of the Study:

To investigate a modified PCR-SSP method for enhanced reliability in ABO blood group typing.
To reduce pseudo-positive signals in PCR-SSP by incorporating mismatched nucleotides into primers.

Main Methods:

Genomic DNA was extracted from nail clippings of 27 volunteers.
PCR-SSP was performed using primers with and without mismatched nucleotides at the 3'-end.
Results were compared with serological ABO blood group analysis.

Main Results:

The modified PCR-SSP method with mismatched primers yielded ABO typing results consistent with serological analysis.
Standard PCR-SSP without mismatched primers produced observable pseudo-positive signals.
The introduction of mismatched nucleotides effectively suppressed pseudo-positive signals.

Conclusions:

The modified PCR-SSP technique, incorporating mismatched nucleotides, offers a more reliable approach for forensic ABO blood typing.
This method enhances accuracy by mitigating errors caused by pseudo-positive signals.
The study demonstrates the potential of this optimized PCR-SSP for forensic applications.