Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs

Hyo Yong Kim, Taihua Li, Cheulhee Jung, Rongzhan Fu, Dae Yeon Cho, Ki Soo Park, Hyun Gyu Park

Research output: Contribution to journalArticle

Abstract

We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5′-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5′-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost.

Original languageEnglish
Pages (from-to)37391-37395
Number of pages5
JournalRSC Advances
Volume8
Issue number65
DOIs
Publication statusPublished - 2018 Jan 1
Externally publishedYes

Fingerprint

Fluorescence
DNA Probes
DNA
Nucleic acids
Nucleic Acids
Assays
Pathogens
Conformations
Costs
Hydrolysis
pyrrolo-deoxycytidine

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)

Cite this

Kim, H. Y., Li, T., Jung, C., Fu, R., Cho, D. Y., Park, K. S., & Park, H. G. (2018). Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs. RSC Advances, 8(65), 37391-37395. https://doi.org/10.1039/C8RA06675B

Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs. / Kim, Hyo Yong; Li, Taihua; Jung, Cheulhee; Fu, Rongzhan; Cho, Dae Yeon; Park, Ki Soo; Park, Hyun Gyu.

In: RSC Advances, Vol. 8, No. 65, 01.01.2018, p. 37391-37395.

Research output: Contribution to journalArticle

Kim, HY, Li, T, Jung, C, Fu, R, Cho, DY, Park, KS & Park, HG 2018, 'Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs', RSC Advances, vol. 8, no. 65, pp. 37391-37395. https://doi.org/10.1039/C8RA06675B
Kim, Hyo Yong ; Li, Taihua ; Jung, Cheulhee ; Fu, Rongzhan ; Cho, Dae Yeon ; Park, Ki Soo ; Park, Hyun Gyu. / Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs. In: RSC Advances. 2018 ; Vol. 8, No. 65. pp. 37391-37395.
@article{8d0f541523f743c8baa9f5a51d171696,
title = "Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs",
abstract = "We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5′-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5′-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost.",
author = "Kim, {Hyo Yong} and Taihua Li and Cheulhee Jung and Rongzhan Fu and Cho, {Dae Yeon} and Park, {Ki Soo} and Park, {Hyun Gyu}",
year = "2018",
month = "1",
day = "1",
doi = "10.1039/C8RA06675B",
language = "English",
volume = "8",
pages = "37391--37395",
journal = "RSC Advances",
issn = "2046-2069",
publisher = "Royal Society of Chemistry",
number = "65",

}

TY - JOUR

T1 - Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs

AU - Kim, Hyo Yong

AU - Li, Taihua

AU - Jung, Cheulhee

AU - Fu, Rongzhan

AU - Cho, Dae Yeon

AU - Park, Ki Soo

AU - Park, Hyun Gyu

PY - 2018/1/1

Y1 - 2018/1/1

N2 - We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5′-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5′-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost.

AB - We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties. In addition, the second, signal-on method is devised by designing the primers to contain 5′-overhang sequences complementary to the PdC-incorporated DNA probes. At the initial phase, the PdC-incorporated DNA probes are hybridized to the 5′-overhang sequences of the primer, exhibiting the significantly quenched fluorescence signal, but are detached by either hydrolysis or strand displacement reaction during PCR, leading to the highly enhanced fluorescence signal. This method is more advanced than the first one since it produces signal-on fluorescence response and permits the use of a single PdC-incorporated DNA probe for the detection of multiple target nucleic acids, remarkably decreasing the assay cost. With these novel qPCR methods, we successfully quantified target nucleic acids derived from sexually transmitted disease (STD) pathogens with high accuracy. Importantly, the proposed strategies overcome the major drawbacks in the current SYBR Green and TaqMan probe-based qPCR methods such as low specificity and high assay cost.

UR - http://www.scopus.com/inward/record.url?scp=85056755986&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85056755986&partnerID=8YFLogxK

U2 - 10.1039/C8RA06675B

DO - 10.1039/C8RA06675B

M3 - Article

VL - 8

SP - 37391

EP - 37395

JO - RSC Advances

JF - RSC Advances

SN - 2046-2069

IS - 65

ER -