Simultaneous Amplification and Detection of Specific DNA Sequences

Higuchi, Russell; Dollinger, Gavin; Walsh, P. Sean; Griffith, Robert L.

doi:10.1038/nbt0492-413

Cited by 880 publications

(460 citation statements)

References 24 publications

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“…5,6 In this study we have developed a related system using fluorescent hybridization probes in the LightCycler. 8,30 One advantage of this system is the rapid cycling time (Ͻ40 s/cycle) due to amplification being performed in glass capillaries and being driven by forced air heating. This system enables the temperature in each reaction to be changed very rapidly.…”

Section: Figurementioning

confidence: 99%

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

et al. 1999

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We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 10 5 cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-␣ (n ‫؍‬ 219), allogeneic BMT (n ‫؍‬ 17), chemotherapy (n ‫؍‬ 11), or at diagnosis (n ‫؍‬ 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n ‫؍‬ 201, r ‫؍‬ 0.90, P Ͻ 0.0001); (2) the proportion of Philadelphia chromosome positive metaphases determined by cytogenetics (n ‫؍‬ 81, P Ͻ 0.0001); and (3) the BCR ratio determined by Southern blot analysis (n ‫؍‬ 122, P Ͻ 0.0001). We conclude that real-time PCR with hybridization probes is a reliable and sensitive method to monitor CML patients after therapy. The major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete PCR analysis takes less than 60 min.

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Section: Figurementioning

confidence: 99%

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

et al. 1999

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“…In order to monitor fluorescence emanating from a PCR, a bifurcated fiber-optic cable was first used to both deliver excitation light from a spectrofluorometer to the PCR and to send emmitted light back to the spectrofluorometer (Higuchi, et al, 1992). A fluorescence-monitoring, thermalcycling instrument capable of spectral analysis and based on fiber-optic transmission and a laser light source is now commercially available (Applied Biosystems Model 7700; Perkin-Elmer, Norwalk, CT).…”

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confidence: 99%

Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes

Higuchi

Watson

1999

PCR Applications

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“…In general, these methods have been based on end-point or competitive PCR analyses measuring band intensities in ethidium bromide stained gels or hybridization-based approaches using radioisotopic labeling and densitometry. [23][24][25][26] Higuchi et al 27,28 introduced fluorescence monitoring at each cycle for quantitative PCR analysis using ethidium bromide to monitor DNA synthesis. Since then, real-time PCR has gained increasing application in molecular diagnostics.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence PCR Quantification of Cyclin D1 Expression

Elenitoba-Johnson

Bohling

Jenson

et al. 2002

The Journal of Molecular Diagnostics

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We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n ‫؍‬ 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n ‫؍‬ 11), acute lymphoblastic leukemia/lymphoma (n ‫؍‬ 15), follicular lymphoma (n ‫؍‬ 6), peripheral T-cell lymphoma (PTCL) (n ‫؍‬ 3), anaplastic large cell lymphoma (n ‫؍‬ 3), hairy cell leukemia (n ‫؍‬ 3), Burkitt lymphoma (n ‫؍‬ 1), Burkitt-like lymphoma (n ‫؍‬ 4), and plasmacytoma (n ‫؍‬ 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A ␤-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma , small cell carcinoma , and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple , rapid , and accurate technique for assessing cyclin D1 expression , and while it is not specific , it can reliably be Mantle cell lymphoma (MCL) is a distinct clinicopathologic entity that is characterized by the presence of the t(11;14)(q13;q32) chromosomal translocation. 1-3 The t(11;14) results in juxtaposition of the bcl-1 locus in close proximity to the immunoglobulin heavy chain enhancer, resulting in deregulation and overexpression of cyclin D1, an important regulator of G1/S progression in the cell cycle. 4,5 MCL shares some histological and immunophenotypic features with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and other low-grade Bcell lymphomas. Because MCL is a clinically aggressive neoplasm, its distinction from CLL/SLL and other lowgrade B-cell lymphomas is important. In this regard, the detection of the t(11;14) has served as a good discriminator of MCL from other entities exhibiting similar histopathological features. The translocation can be detected in 90 to 95% of MCLs by fluorescence in situ hybridization, 6 70 to 80% by conventional cytogenetics, 7 60 to 70% by Southern blot hybridization, 8 and 30 to 40% using polymerase chain reaction (bcl-1, major translocation cluster/immunoglobulin joining). 9,10 On the other hand, cyclin D1 protein expression is demonstrable in approximatel...

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Simultaneous Amplification and Detection of Specific DNA Sequences

Cited by 880 publications

References 24 publications

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes

Fluorescence PCR Quantification of Cyclin D1 Expression

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