Time-resolved fluorometry in end-point and real-time PCR quantification of nucleic acids
Nurmi, Jussi ; Lilja, Hans LU
and Ylikoski, Alice
(2000)
In Luminescence
15(6).
p.381-388
- Abstract
Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5′→3′ exonucleolytic DNA polymerase and a... (More)
Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5′→3′ exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 × 106 and 10-5 × 107 input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.
(Less)
- author
- Nurmi, Jussi
; Lilja, Hans
LU
and Ylikoski, Alice
- organization
- publishing date
- 2000
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Heterogeneous hybridization, Prostate-specific antigen, Quantitative PCR, Real-time detection, Time-resolved fluorometry
- in
- Luminescence
- volume
- 15
- issue
- 6
- pages
- 381 - 388
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:11114115
- scopus:0034328582
- ISSN
- 1522-7235
- DOI
- 10.1002/1522-7243(200011/12)15:6<381::AID-BIO623>3.0.CO;2-3
- language
- English
- LU publication?
- yes
- id
- 98bcc297-7b87-4e6f-a7bd-00f2dafd5f46
- date added to LUP
- 2022-12-06 17:32:15
- date last changed
- 2024-01-13 13:21:02
@article{98bcc297-7b87-4e6f-a7bd-00f2dafd5f46,
abstract = {{<p>Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5′→3′ exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 × 10<sup>6</sup> and 10-5 × 10<sup>7</sup> input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.</p>}},
author = {{Nurmi, Jussi and Lilja, Hans and Ylikoski, Alice}},
issn = {{1522-7235}},
keywords = {{Heterogeneous hybridization; Prostate-specific antigen; Quantitative PCR; Real-time detection; Time-resolved fluorometry}},
language = {{eng}},
number = {{6}},
pages = {{381--388}},
publisher = {{John Wiley & Sons Inc.}},
series = {{Luminescence}},
title = {{Time-resolved fluorometry in end-point and real-time PCR quantification of nucleic acids}},
url = {{http://dx.doi.org/10.1002/1522-7243(200011/12)15:6<381::AID-BIO623>3.0.CO;2-3}},
doi = {{10.1002/1522-7243(200011/12)15:6<381::AID-BIO623>3.0.CO;2-3}},
volume = {{15}},
year = {{2000}},
}