Rapid ABO Genotyping Using Whole Blood without DNA Purification

Sung Ho Lee; Geon Park; Young Geun Yang; Seung Gwan Lee; Suhng Wook Kim

doi:10.3343/kjlm.2009.29.3.231

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Lee, Park, Yang, Lee, and Kim: Rapid ABO Genotyping Using Whole Blood without DNA Purification

Original Article

Korean J Leg Med 2009;29(3):231-237.

Published online: 14 January 2009

DOI: https://doi.org/10.3343/kjlm.2009.29.3.231

Rapid ABO Genotyping Using Whole Blood without DNA Purification

Sung Ho Lee, Ph.D.¹, Geon Park, M.D.², Young Geun Yang, Ph.D.³, Seung Gwan Lee, Ph.D.⁴, Suhng Wook Kim, Ph.D.⁴

¹R&D Center, Lumieye Genetics Co., Ltd., Seoul, Korea

²Department of Laboratory Medicine, Chosun University College of Medicine, Gwangju, Korea

³BioQuest, Inc., Seoul, Korea

⁴Department of Clinical Laboratory Science, College of Health Sciences, Korea University, Seoul, Korea

Corresponding author: Suhng Wook Kim, Ph.D. Department of Clinical Laboratory Science, College of Health Sciences, Korea University, San-1 Jeongreung-dong, Seongbuk-gu, Seoul 136-703, Korea Tel: +82-2-940-2811, Fax: +82-2-917-2388 E-mail: swkimkorea@korea.ac.kr

Revised 21 July 200823 April 2009 Accepted 23 April 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification.

Methods:

A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method.

Results:

The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies.

Conclusions:

This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.

Keywords: ABO genotyping, Whole blood, Allele-specific polymerase chain reaction

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Fig. 1.

Primer positions for ABO genotyping. The key polymorphisms are indicated at positions, 261, 297, 467, and 803. Alleles O01 and O02 have a G deletion at nucleotide position 261 (the next nucleotide is shown in parentheses). Nucleotides at these positions were detected in PCR reactions using the appropriate primers as shown with arrows (given in Table 1).

Fig. 2.

Electropherogram of selected ABO genotypes. The gel photograph showing the size difference of the amplified products was obtained from the samples with 13 independent genotypes. The internal control produces a distinct 500 bp band in all four reactions. Lane M, size marker (100-500 bp ladder); lane 1, PCR reaction 1; lane 2, PCR reaction 2; lane 3, PCR reaction 3; lane 4, PCR reaction 4.

Table 1.

Primers used for ABO genotyping

PCR reaction	Primer pair	Fragment size (bp)	Allele specificity
1	261G: 5′-GCAGTAGGAAGGATGTCCTCGTGtTG-3′	205	A101, A102, B101, cis-AB01
	int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′
	467C: 5′-CCACTACTATGTCTTCACCGACCAtCC-3′	381	A101, O01, O02
	803G: 5′-CACCGACCCCCCGAAGAtCC-3′
2	297A: 5′-CCATTGTCTGGGAGGGCcCA-3′	164	A101, A102, O01, cis-AB01
	int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′
	467C: 5′-CCACTACTATGTCTTCACCGACCAtCC-3′	381	B101
	803C: 5′-CACCGACCCCCCGAAGAtCG-3′
3	261A: 5′-GCAGTAGGAAGGATGTCCTCGTGtTA-3′	205	O01, O02
	int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′
	467T: 5′-CCACTACTATGTCTTCACCGACCAtCT-3′	381	A102
	803G: 5′-CACCGACCCCCCGAAGAtCC-3′
4	297G: 5′-CCATTGTCTGGGAGGGCcCG-3′	164	B101, O02
	int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′
	467T: 5′-CCACTACTATGTCTTCACCGACCAtCT-3′	381	cis-AB01
	803C: 5′-CACCGACCCCCCGAAGAtCG-3′

The primers are named based on the position of their 3' end relative to the cDNA sequence of A101 allele. The nucleotides not complementary with the target template are in lower case.

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