A homogeneous, ligase-mediated DNA diagnostic test.
Journal: 1998/August - Genome Research
ISSN: 1088-9051
PUBMED: 9582198
Abstract:
Single-nucleotide variations are the most widely distributed genetic markers in the human genome. A subset of these variations, the substitution mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecular diagnosis of genetic disorders and large-scale population studies for genetic analysis of complex traits, a simple, sensitive, and specific test for single nucleotide changes is highly desirable. In this report we describe the development of a homogeneous DNA detection method that requires no further manipulations after the initial reaction is set up. This assay, named dye-labeled oligonucleotide ligation (DOL), combines the PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection monitored in real time. Because FRET occurs only when the donor and acceptor dyes are in close proximity, one can infer the genotype or mutational status of a DNA sample by monitoring the specific ligation of dye-labeled oligonucleotide probes. We have successfully applied the DOL assay to genotype 10 SNPs or mutations. By designing the PCR primers and ligation probes in a consistent manner, multiple assays can be done under the same thermal cycling conditions. The standardized design and execution of the DOL assay means that it can be automated for high-throughput genotyping in large-scale population studies.
Relations:
Content
Citations
(17)
References
(15)
Diseases
(1)
Chemicals
(5)
Organisms
(1)
Processes
(2)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Genome Res 8(5): 549-556

A Homogeneous, Ligase-Mediated DNA Diagnostic Test

Division of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110 USA; Perkin-Elmer Applied Biosystems Division, Foster City, California 94404 USA
Corresponding author.
Received 1998 Jan 29; Accepted 1998 Mar 2.

Abstract

Single-nucleotide variations are the most widely distributed genetic markers in the human genome. A subset of these variations, the substitution mutations, are responsible for most genetic disorders. As single nucleotide polymorphism (SNP) markers are being developed for molecular diagnosis of genetic disorders and large-scale population studies for genetic analysis of complex traits, a simple, sensitive, and specific test for single nucleotide changes is highly desirable. In this report we describe the development of a homogeneous DNA detection method that requires no further manipulations after the initial reaction is set up. This assay, named dye-labeled oligonucleotide ligation (DOL), combines the PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection monitored in real time. Because FRET occurs only when the donor and acceptor dyes are in close proximity, one can infer the genotype or mutational status of a DNA sample by monitoring the specific ligation of dye-labeled oligonucleotide probes. We have successfully applied the DOL assay to genotype 10 SNPs or mutations. By designing the PCR primers and ligation probes in a consistent manner, multiple assays can be done under the same thermal cycling conditions. The standardized design and execution of the DOL assay means that it can be automated for high-throughput genotyping in large-scale population studies.

Abstract

The ability to determine efficiently and unambiguously the mutational status or genotype of any living organism has great applications in molecular diagnostics, clinical genetic testing, population genetics, and agricultural biotechnology (Risch and Merikangas 1996; Collins et al. 1997). High-throughput detection methods for single-nucleotide variations currently in use include homogeneous methods such as the template-directed dye-terminator incorporation (TDI) assay (Chen and Kwok 1997), the 5′-nuclease allele-specific hybridization TaqMan assay (Livak et al. 1995b), and the recently described allele-specific molecular beacon assay (Tyagi et al. 1998). The TDI assay combines PCR and specific primer extension with fluorescence resonance energy transfer (FRET) detection and requires almost no optimization, but it requires three separate steps that include adding reagents twice after the initial reaction setup (Chen et al. 1997). The TaqMan assay is the simplest of all diagnostic assays in which one can determine the mutational status of a DNA sample in one step during PCR (Livak et al. 1995b). However, the TaqMan assay has the stringent requirement that a perfectly complementary internal probe hybridizes and is cleaved at the polymorphic site during the strand extension phase in PCR, whereas the corresponding probe with a 1-base mismatch does not. This requires some optimization and may be difficult to achieve in AT-rich domains. Allele discrimination by use of molecular beacons of four different colors has been shown to work very well when synthetic oligonucleotides are used as targets, but assay conditions for amplified or genomic DNA targets are still being worked out (Tyagi et al. 1998).

Here, we report the development of the DOL assay that has all the desirable properties of a homogeneous DNA detection test. It combines the highly sensitive and specific PCR–OLA (oligonucleotide ligation assay) with FRET detection in one reaction in which the mutational status or genotype is determined without any purification or manipulation after the reaction is set up initially. FRET is observed when two fluorescent dyes are in close proximity and the donor dye’s emission spectrum overlaps the acceptor dye’s excitation spectrum. When the donor is excited, the donor’s specific emission decreases (quenching) with a concomitant increase in the acceptor’s specific emission (Foster 1965). Although the efficiency of FRET is very sensitive to the distance between the donor and acceptor, with energy transfer dropping dramatically as the distance between them increases, the hydrophobic interactions between the dyes make it possible to observe FRET even when the donor and acceptor are placed >30 bases apart on an oligonucleotide in an aqueous solution (Livak et al. 1995a; Chen and Kwok 1997) The ability to detect intramolecular FRET against the background of unquenched donor emission provides a detection system that requires no separation or purification of the product in the DOL assay. The changes in fluorescence intensities during the ligation reaction can be monitored in real time by a fluorescence spectrophotometer connected to a thermal cycler, or at its end point by means of a fluorescence plate reader.

The experimental design of this study is shown in Figure Figure1.1. PCR primers are designed to have high melting temperatures (Tms) with the aid of a primer selection program. Three dye-labeled ligation probes are needed for each biallelic marker. A 5′-donor dye-labeled common probe is designed to terminate one base immediately upstream from the polymorphic site. Two allele-specific, 5′-phosphorylated, 3′-acceptor dye-labeled probes are designed to have the allelic base at the 5′-end. All three ligation probes are designed with the same primer selection program to have matching but low Tms. The donor dye used in this study is FAM (5-carboxy-fluorescein), the acceptor dyes used are ROX (6-carboxy-X-rhodamine) and TAMRA (N,N,N′,N′-tetramethyl-6-carboxyrhodamine). A thermostable DNA polymerase with no 5′-nuclease activity (AmpliTaq, FS) and a thermostable DNA ligase (Ampligase) are used. The genomic DNA test sample is added to the reaction mixture containing the two PCR primers, three ligation probes, AmpliTaq, FS DNA polymerase, Ampligase, and a reaction buffer containing nicotinamide–adenine dinucleotide (NAD). Thermal cycling is performed in a thermal cycler equipped with a spectrophotometer such that fluorescence intensities of the donor and acceptor dyes can be monitored in real time. During the first (PCR) stage of the reaction, the annealing/extension temperature is kept high (∼65°C) such that the ligation probes are unable to anneal. This prevents the extension of the FAM-labeled ligation probe during the PCR phase of the reaction. After sufficient PCR products have been formed, the second (ligation) stage of the reaction is done with low annealing/ligation temperature (∼45°C) such that the ligation probes can anneal and be ligated together. The use of a DNA polymerase (AmpliTaq, FS) lacking 5′-nuclease activity prevents the cleavage of ligation probes during the ligation phase of the reaction.

An external file that holds a picture, illustration, etc.
Object name is gr.5f1.jpg

DOL assay with FRET detection. All of the reagents, including DNA polymerase, DNA ligase, PCR primers, and ligation probes, are added to the test DNA sample when the reaction is first set up. In a two-stage thermal cycling sequence, PCR is allowed to proceed without the ligation probe annealing to the target followed by the ligation reaction when enough PCR product has accumulated. At the end of the assay, there are four possible outcomes for each test sample: (A) homozygous for the first allele leading to the formation of a ligation product containing the G allele in this example in the absence of the ligation product corresponding to the second allele (A allele in this example); (B) heterozygous with ligation product of both alleles formed; (C) homozygous for the second allele with the ligation product containing the A allele formed in this example in the absence of the G allele ligation product; (D) PCR or ligation failure leading to no ligation product being formed. (Blue) FAM; (green) TAMRA; (red) ROX.

Acknowledgments

We thank G. Pilia and B. Zehnbauer for DNA samples, S. Downey and B. Mullah for ligation probe synthesis, R. Jones and E. Shulse for careful reading of the manuscript. This work was supported in part by grants from the National Institutes of Health.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Footnotes

E-MAIL ude.ltsuw.mi@kowk; FAX (314) 362-8159.

Footnotes

REFERENCES

REFERENCES
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.