ES2545577T3 - Método mejorado para el tratamiento con bisulfito - Google Patents

Método mejorado para el tratamiento con bisulfito Download PDF

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Publication number
ES2545577T3
ES2545577T3 ES07012188.4T ES07012188T ES2545577T3 ES 2545577 T3 ES2545577 T3 ES 2545577T3 ES 07012188 T ES07012188 T ES 07012188T ES 2545577 T3 ES2545577 T3 ES 2545577T3
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Prior art keywords
bisulfite
dna
solid phase
glass
nucleic acid
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Christine Dr. Markert-Hahn
Dirk Dr. Block
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Epigenomics AG
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Epigenomics AG
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Priority claimed from EP02019097A external-priority patent/EP1394172A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Compounds Of Iron (AREA)
  • Saccharide Compounds (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

Método para la conversión de una base citosina en un ácido nucleico a una base uracilo, que comprende las etapas de: a) incubar el ácido nucleico en presencia de iones sulfito de manera que el ácido nucleico se desamina, b) unir el ácido nucleico desaminado a una fase sólida, c) lavar opcionalmente el ácido nucleico desaminado unido a la fase sólida, d) incubar el ácido nucleico unido a la fase sólida desaminado bajo condiciones alcalinas de manera que el ácido nucleico desaminado se desulfona, e) lavar opcionalmente el ácido nucleico unido a la fase sólida desaminado y desulfonado, y f) eluir opcionalmente el ácido nucleico desaminado y desulfonado a partir de la fase sólida.

Description

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1.2 Metodología General
El experimento siguiente demuestra que la PCR que se describe en el instrumento LightCycler® puede utilizarse como herramienta de evaluación del ADN que se trató con bisulfito. Muestra que la combinación iniciador/sonda diseñada da resultados positivos solamente con ADN después del tratamiento con bisulfito. El ADN tratado con bisulfito (en este caso el ADN se trató con bisulfito de acuerdo con el protocolo que se describe en el Ejemplo 2) y el ADN no tratado se amplificaron en paralelo con la utilización de las mismas concentraciones de molde (20ng y 1ng por PCR).
1.3 Análisis por PCR en el Instrumento LightCycler® 1.3.1 Composición de mezcla madre:
Sonda de hibridación LC FastStart DNA Master Hybridation Probe 1x, MgCl2 2 mM, iniciador directo 0.5μM,iniciador inverso 0.5μM, sonda donadora 250nM, sonda aceptora 250nM, molde 10μl, volumen total de la PCR 20μl.
1.3.2 Condiciones de la PCR:
Desnaturalización 10min/95°C 55 ciclos 95°C/10s 65°C/10s - captación de señal 72°C110s Incremento gradual de la temperatura 20°C/s
1.4 Resultado
MDNA/PCR
Tratamiento con bisulfito Valor de CT o Punto de cruce
imagen11
imagen12 imagen13
20 ng
Sí 30.55
imagen14
imagen15 29.72
imagen16
imagen17 29.95
imagen18
imagen19 30.06
1 ng
Sí 34.7
imagen20
imagen21 35.8
imagen22
imagen23 34.07
imagen24
imagen25 33.86
20 ng
No Sin curva de crecimiento
imagen26
imagen27 Sin curva de crecimiento
imagen28
imagen29 Sin curva de crecimiento
imagen30
imagen31 Sin curva de crecimiento
1 ng
No Sin curva de crecimiento
imagen32
imagen33 Sin curva de crecimiento
imagen34
imagen35 Sin curva de crecimiento
imagen36
imagen37 Sin curva de crecimiento
El resultado muestra puntos de cruce solamente para el ADN que se trató con bisulfito. Por lo tanto, esta PCR es adecuada para evaluar los métodos con bisulfito. Para aquellos con experiencia en la materia está claro que podría utilizarse cualquier PCR como herramienta de evaluación si se garantiza que la combinación iniciador/sonda no reacciona con el ADN antes del tratamiento con bisulfito.
2. Ejemplo 2: Reacción del bisulfito mediante la utilización de partículas de vidrio magnéticas (MGPs)
2.1.1 Desnaturalización del ADN:
12
imagen38
2.4.3 Resultados:
imagen39
ADN metilado por Método del Bisulfito que se utilizó
réplicas
Bisulfito PCR Intergen Método MGP
imagen40
imagen41 imagen42 Valores de CT o Puntos de cruce
1
30 ng 6 ng 29.90 30.46
2
imagen43 imagen44 30.07 29.86
3
imagen45 imagen46 30.07 30.44
4
imagen47 imagen48 30.14 30.35
5
imagen49 imagen50 30.22 30.24
6
imagen51 imagen52 30.26 30.46
7
imagen53 imagen54 30.31 30.50
8
imagen55 imagen56 30.19 30.54
9
imagen57 imagen58 30.03 30.17
10
imagen59 imagen60 29.85 30.69
1
6ng 1.2 ng 32.49 32.14
2
imagen61 imagen62 32.67 32.60
3
imagen63 imagen64 32.29 32.83
4
imagen65 imagen66 32.87 32.53
5
imagen67 imagen68 32.15 32.90
6
imagen69 imagen70 32.23 32.77
7
imagen71 imagen72 32.59 32.73
8
imagen73 imagen74 32.91 33.09
9
imagen75 imagen76 32.46 32.88
10
imagen77 imagen78 33.17 32.83
Los valores de CT o puntos de cruce que se calculan durante la PCR en tiempo real son casi idénticos para los dos métodos con bisulfito que se utilizaron, es decir que el desempeño de los métodos es el mismo.
3 Ejemplo 3: Reacción con bisulfito automatizada mediante la utilización de MGPs
3.1 Realización de la reacción con bisulfito: 3.1.1 Desnaturalización del ADN:
Se mezclan y se incuban durante 10 minutos a 37 °C 20 μl de la dilución de ADN metilado (Intergen, que se distribuye por Serologicals Corporation, Norcross, GA, Estados Unidos; Cat S7821) (50 ng/ensayo), 4 μl de una solución de Poli(dA)(concentración 250 ng/ μl) y2.6 μl de NaOH 2 M.
3.1.2 Desaminación de ADN
Se mezclan 26 μl de ADN desnaturalizado con 220 μl de reactivo de bisulfito (bisulfito de sodio 2.5M, hidroquinona125mM, pH 5.0) y se incuban durante 4 horas a 50 °C.
3.1.3 Procesamiento automatizado mediante la utilización del instrumento LC MagNAPure
Se mezclan 250 μl de ADN desaminado con 600 μl de amortiguador de unión (estuche de aislamiento de ADN MagNAPureDNA Isolation Kit I, Roche, Mannheim, Alemania) y 75 μl de la solución de partículas de vidrio magnéticas (estuche de aislamiento de ADN MagNAPure DNA Isolation Kit I, Roche, Mannheim, Alemania) y se incuban durante 15min/ temperatura ambiente con mezclado continuo. Después de eso, las partículas de vidrio magnéticas se lavan tres
14
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imagen80
imagen81
Lista de Referencias
Abramson, R. D. y Myers, T. W., Curr Opin Biotechnol 4 (1993) 41-7 Alderton, R. P., y otros, Anal Biochem 201 (1992) 166-9 Ausubel, F., y otros, en "Current protocols in molecular biology" (1994), Eds. F. Ausubel, R. Brent y K. R.E., Wiley & Sons Verlag, Nueva York Barany, F., PCR Methods Appl 1 (1991) 5-16 Barany, F., Proc Natl Acad Sci U S A 88 (1991) 189-93 Benyajati, C., y otros, Nucleic Acids Res 8 (1980) 5649-67 Braunauer, en "The Adsorption of Gases and Vapors" (1943), Princeton University Press Clark, S. J., y otros, Nucleic Acids Res 22 (1994) 2990-7 DE 3724442 DE-A 37 34 442 EP 0 200 362 EP 0 201 184 EP 0 389 063 EP 0 439 182 Feil, R., y otros, Nucleic Acids Res 22 (1994) 695-6 Frommer, M., y otros, Proc Natl Acad Sci U S A 89 (1992) 1827-31 GB 91/00212 Grigg, G. y Clark, S., Bioessays 16 (1994) 431-6 Grigg, G. W., DNA Seq 6 (1996) 189-98 Grunau, C., y otros, Nucleic Acids Res 29 (2001) E65-5 Guatelli, J. C., y otros, Proc Natl Acad Sci U S A 87 (1990) 1874-8 Jakobi, R., y otros, Anal Biochem 175 (1988) 196-201 Komiyama, M. y Oshima, S., Tetrahedron Letters 35 (1994) 8185-8188 Kwoh, D. Y., y otros, Proc Natl Acad Sci U S A 86 (1989) 1173-7 Lottspeich y Zorbas, en "Bioanalytik" (1998), Eds. L. a. Zorbas, Spektrum Akademischer Verlag, Heidelberg, Berlín, Alemania Marko, M. A., y otros, Anal Biochem 121 (1982) 382-7 Morrow, C.S., y otros, Gene 75 (1989), 3-11 Oakeley, E. J., Pharmacol Ther 84 (1999) 389-400 Olek, A., y otros, Nucleic Acids Res 24 (1996) 5064-6 Paulin, R., y otros, Nucleic Acids Res 26 (1998) 5009-10 Raizis, A. M., y otros, Anal Biochem 226 (1995) 161-6 Sambrook, J., y otros, en "Molecular Cloning: A Laboratory Manual" (1989), Eds. J. Sambrook, E. F. Fritsch y T. Maniatis, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY Spray Drying Handbook (1991), John Wiley & Sons, Nueva York US 4,683,202 US 5,130,238 US 5,137,806 US 5,210,015 US 5,234,809 US 5,487,972 US 5,552,277 US 5,595,890 US 5,639,611 US 5,786,146 US 5,804,375 US 6,174,670 US 6,331,393 Vogelstein, B. y Gillespie, D., Proc Natl Acad Sci U S A 76 (1979) 615-9 Warnecke, P. M., y otros, Methods 27 (2002) 101-7 Whelen, A. C. y Persing, D. H., Annu Rev Microbiol 50 (1996) 349-73 WO 00/32762 WO 00/37291 WO 01/37291 WO 01/98528 WO 02/31186 WO 90/01069 WO 90/06045 WO 92/02638
18
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Claims (1)

  1. imagen1
    imagen2
ES07012188.4T 2002-08-29 2003-08-27 Método mejorado para el tratamiento con bisulfito Expired - Lifetime ES2545577T3 (es)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP02019097 2002-08-29
EP02019097A EP1394172A1 (en) 2002-08-29 2002-08-29 Improved method for bisulfite treatment
EP02028114 2002-12-18
EP02028114 2002-12-18

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ES03019321T Expired - Lifetime ES2294231T3 (es) 2002-08-29 2003-08-27 Metodo mejorado para tratamiento por bisulfito.

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EP (2) EP1829886B1 (es)
JP (2) JP5153048B2 (es)
CN (1) CN1271081C (es)
AT (1) ATE374782T1 (es)
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CA (1) CA2438327C (es)
CY (1) CY1107118T1 (es)
DE (1) DE60316642T2 (es)
DK (2) DK1394173T3 (es)
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HK (1) HK1062688A1 (es)
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EP1394173A1 (en) 2004-03-03
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CN1271081C (zh) 2006-08-23
DK1394173T3 (da) 2008-02-04
ES2294231T3 (es) 2008-04-01
CA2438327A1 (en) 2004-02-29
US20160068888A1 (en) 2016-03-10
HK1062688A1 (en) 2004-11-19
CA2438327C (en) 2008-04-15
SI1829886T1 (sl) 2015-12-31
US20040241704A1 (en) 2004-12-02
DE60316642D1 (de) 2007-11-15
AU2003236461B2 (en) 2009-05-28
EP1394173B1 (en) 2007-10-03
JP5310059B2 (ja) 2013-10-09
US9394332B2 (en) 2016-07-19
SI1394173T1 (sl) 2008-02-29
JP2009106304A (ja) 2009-05-21
ATE374782T1 (de) 2007-10-15
DK1829886T3 (en) 2015-08-17
PT1394173E (pt) 2007-11-28
CN1495192A (zh) 2004-05-12
EP1394173B9 (en) 2008-10-15
CY1107118T1 (el) 2012-10-24
HUE027240T2 (en) 2016-10-28
DE60316642T2 (de) 2008-01-31
AU2003236461A1 (en) 2004-03-18
US9868756B2 (en) 2018-01-16
JP5153048B2 (ja) 2013-02-27
EP1829886A2 (en) 2007-09-05
EP1829886A3 (en) 2008-10-15

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