Unlocked nucleic acid modified primer-based enzymatic polymerization assay: Towards allele-specific genotype detection of human platelet antigens

Bao T. Le, Quintin Hughes, Shilpa Rakesh, Ross Baker, Per T. Jørgensen, Jesper Wengel*, Rakesh N. Veedu

*Corresponding author for this work

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Abstract

Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.

Original languageEnglish
JournalRSC Advances
Volume8
Issue number57
Pages (from-to)32770-32774
ISSN2046-2069
DOIs
Publication statusPublished - 2018

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Human Platelet Antigens
Nucleic acids
Antigens
Nucleotides
Platelets
Polymorphism
Nucleic Acids
Assays
Polymerization
DNA
Nucleic Acid Probes
Polymerase chain reaction
DNA-Directed DNA Polymerase

Cite this

@article{17c77c3b0d884487a8015382f86696d5,
title = "Unlocked nucleic acid modified primer-based enzymatic polymerization assay: Towards allele-specific genotype detection of human platelet antigens",
abstract = "Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.",
author = "Le, {Bao T.} and Quintin Hughes and Shilpa Rakesh and Ross Baker and J{\o}rgensen, {Per T.} and Jesper Wengel and Veedu, {Rakesh N.}",
year = "2018",
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Unlocked nucleic acid modified primer-based enzymatic polymerization assay : Towards allele-specific genotype detection of human platelet antigens. / Le, Bao T.; Hughes, Quintin; Rakesh, Shilpa; Baker, Ross; Jørgensen, Per T.; Wengel, Jesper; Veedu, Rakesh N.

In: RSC Advances, Vol. 8, No. 57, 2018, p. 32770-32774.

Research output: Contribution to journalJournal articleResearchpeer-review

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AU - Le, Bao T.

AU - Hughes, Quintin

AU - Rakesh, Shilpa

AU - Baker, Ross

AU - Jørgensen, Per T.

AU - Wengel, Jesper

AU - Veedu, Rakesh N.

PY - 2018

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N2 - Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.

AB - Accurate detection of single nucleotide polymorphisms (SNPs) is paramount for the appropriate therapeutic intervention of debilitating diseases associated with SNPs. However, in some cases current nucleic acid probes fail to detect allele-specific mutations, for example, human platelet antigens, HPA-15a (TCC) and HPA-15b (TAC) alleles associated with neonatal alloimmune thrombocytopenia. Towards this, it is necessary to develop a novel assay for detection of allele-specific mutations. In this study, we investigated the potential of unlocked nucleic acid (UNA)-modified primers in SNP detection utilising an enzymatic polymerisation-based approach. Our results of primer extension and asymmetric polymerase chain reaction by KOD XL DNA polymerase revealed that UNA-modified primers achieved excellent allele-specificity in discriminating the human platelet antigen DNA template, whereas the DNA control primers were not able to differentiate between the normal and mutant alleles, demonstrating the scope of this novel UNA-based enzymatic approach as a robust methodology for efficient detection of allele-specific mismatches. Although further evaluation is required for other disease conditions, we firmly believe that our findings offer a great promise for the diagnosis of neonatal alloimmune thrombocytopenia and other SNP-related diseases.

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