ES2532141T3 - Cebadores de polinucleótidos para detectar mutaciones de PIK3CA - Google Patents
Cebadores de polinucleótidos para detectar mutaciones de PIK3CA Download PDFInfo
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- ES2532141T3 ES2532141T3 ES10158519.8T ES10158519T ES2532141T3 ES 2532141 T3 ES2532141 T3 ES 2532141T3 ES 10158519 T ES10158519 T ES 10158519T ES 2532141 T3 ES2532141 T3 ES 2532141T3
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- 230000035772 mutation Effects 0.000 title abstract description 17
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 title abstract description 3
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 title abstract description 3
- 102000040430 polynucleotide Human genes 0.000 title abstract 5
- 108091033319 polynucleotide Proteins 0.000 title abstract 5
- 239000002157 polynucleotide Substances 0.000 title abstract 5
- 101150063858 Pik3ca gene Proteins 0.000 abstract description 3
- 230000000295 complement effect Effects 0.000 abstract description 3
- 238000000034 method Methods 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 239000012634 fragment Substances 0.000 abstract 2
- 238000009396 hybridization Methods 0.000 abstract 2
- 108020004707 nucleic acids Proteins 0.000 abstract 2
- 102000039446 nucleic acids Human genes 0.000 abstract 2
- 150000007523 nucleic acids Chemical class 0.000 abstract 2
- 238000003556 assay Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 241000239226 Scorpiones Species 0.000 description 11
- 102200085788 rs121913279 Human genes 0.000 description 4
- 102200085789 rs121913279 Human genes 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
Un método de PCR para detectar la presencia o la ausencia de una mutación en el gen PIK3CA que comprende las etapas de: a) mezclar una muestra de ácido nucleico que comprende al menos un fragmento del gen PIK3CA con una secuencia de polinucleótido cebador que consiste en la SEQ ID NO. 14, donde el polinucleótido se usa como cebador y el segundo cebador corresponde a una región del fragmento de la secuencia de PIK3CA por debajo de la región a la cual el polinucleótido es complementario, y llevar a cabo una PCR con la mezcla; y b) detectar la hibridación del polinucleótido a la muestra de ácido nucleico en donde la hibridación indica la presencia de una mutación.
Description
E10158519
05-03-2015
Región de exón 9
- Mutación
- Secuencia de cebador SEQ. ID. NO.
- E542K-0
- 5'-CTTTCTCCTGCTCAGTGATTTT-3' 3
- E542K-1
- 5'-CTTTCTCCTGCTCAGTGATTAT-3' 4
- E542K-2
- 5'-CTTTCTCCTGCTCAGTGATTCT-3' 5
- E542K-3
- 5'-CTTTCTCCTGCTCAGTGATTGT-3' 6
- E542K-4
- 5'-CTTTCTCCTGCTCAGTGATATT-3' 7
- E542K-5
- 5'-CTTTCTCCTGCTCAGTGATCTT-3' 8
- E542K-6
- 5'-CTTTCTCCTGCTCAGTGATGTT-3' 9
- E545K-0
- 5'-ACTCCATAGAAAATCTTTCTCCTGCTT-3' 10
- E545K-1
- 5'-ACTCCATAGAAAATCTTTCTCCTGCAT-3' 11
- E545K-2
- 5'-ACTCCATAGAAAATCTTTCTCCTGCCT-3' 12
- E545K-3
- 5'-ACTCCATAGAAAATCTTTCTCCTGCGT-3' 13
- E545K-4
- 5'-ACTCCATAGAAAATCTTTCTCCTGATT-3' 14
- E545K-5
- 5'-ACTCCATAGAAAATCTTTCTCCTGGTT-3' 15
- E545K-6
- 5'-ACTCCATAGAAAATCTTTCTCCTGTTT-3' 16
- Scorpion de exón 9
-
imagen5 17 y 18
E10158519
05-03-2015
Región de exón 20
La región diana para las mutaciones H1047R y H1047L se muestra a continuación como la SEQ ID NO. 19 (las bases mutantes se muestran entre corchetes con la variante normal en primer lugar). Los cebadores directos para las mutaciones también se muestran a continuación (SEQ ID NOS. 20 a 33). Para potenciar la especificidad de dichas reacciones, se usaron discordancias de cebador adicionales cercanas al extremo 3' (mostradas subrayadas en las secuencias de cebador). Se usaron los cebadores óptimos (H1047R-1 y H1047L-1) para los experimentos descritos. El cebador Scorpions utilizable con las secuencias de cebador se muestra como las SEQ ID NO. 34 y 35. Las regiones de correspondencia entre el cebador Scorpions y las regiones diana se muestran subrayadas o destacadas de forma idéntica.
- Mutación
- Secuencia de cebador SEQ. ID. NO.
- H1047R-0
- 5'-TGTTGTCCAGCCACCATGAC-3' 20
- H1047R-1
- 5'-TGTTGTCCAGCCACCATGCC-3' 21
- H1047R-2
- 5'-TGTTGTCCAGCCACCATGGC-3' 22
- H1047R-3
- 5'-TGTTGTCCAGCCACCATGTC-3' 23
- H1047R-4
- 5'-TGTTGTCCAGCCACCATAAC-3' 24
- H1047R-5
- 5'-TGTTGTCCAGCCACCATCAC-3' 25
- H1047R-6
- 5'-TGTTGTCCAGCCACCATTAC-3' 26
- H1047L-0
- 5'-TGTTGTCCAGCCACCATGAA-3' 27
- H1047L-1
- 5'-TGTTGTCCAGCCACCATGCA-3' 28
- H1047L-2
- 5'-TGTTGTCGAGCCACCATGGA-3' 29
- H1047L-3
- 5'-TGTTGTCCAGCCACCATGTA-3' 30
- H1047L-4
- 5'-TGTTGTCCAGCCACCATAAA-3' 31
- H1047L-5
- 5'-TGTTGTCCAGCCACCATCAA-3' 32
- H1047L-6
- 5'-TGTTGTCCAGCCACCATTAA-3' 33
- Scorpion de exón 20
-
imagen7 34 y 35
5
10
15
20
25
30
35
40
E10158519
05-03-2015
Cebadores de control
A continuación se muestran los cebadores de control. Las regiones de correspondencia entre el cebador Scorpions y las regiones diana se muestran subrayadas o destacadas de forma idéntica.
- Mutación
- Secuencia de cebador SEQ. ID. NO.
- Cebador de control
- 5'-AGATGATCTCATTGTTCTGAAACAG-3' 37
- Scorpion de control
-
imagen9 38 y 39
Todos los cebadores fueron sintetizados y suministrados por Invitrogen. El tampón de PCR, la Taq y el magnesio fueron suministrados por Eurogentec y los dNTPS fueron adquiridos a Abgene Ltd. Los Scorpions fueron suministrados y sintetizados por ATDBio.
Los ensayos fueron multiplexados en 2 reacciones que contenían un ensayo de control y 2 ensayos ARMS (1 x exón 9 y 1 x exón 20). Los ensayos fueron llevados a cabo en un volumen de reacción de 25 µL que contenía 1 x tampón de PCR, 4,0 mM de MgCl2, 200 µM de mezcla de dNTPs, 0,25 µM de cada uno de los cebadores (cebador de control y 2 cebadores ARMS) y 0,25 µM de cada Scorpion (Scorpion de control (SEQ ID NO. 38 y 39), Scorpion de exón 20 (SEQ ID NO. 34 y 35) y Scorpion de exón 9 (SEQ ID NO. 17 y 18)). Se añadieron 2,5 uL de plantilla de ADN a cada reacción. Los cebadores H1047R y E542K fueron multiplexados con 2,5 unidades de polimerasa Taq por reacción. Los cebadores H1047L y E545K fueron multiplexados con 3,0 unidades de polimerasa Taq por reacción. El cebador E542K usado fue el E542K-2 (SEQ ID NO. 5). El cebador E545K usado fue el E545K-4 (SEQ ID NO. 14). El cebador H1047R usado fue el H1047R-1 (SEQ ID NO. 21). El cebador H1047L usado fue el H1047L-1 (SEQ ID NO. 28).
En todos los casos las reacciones fueron amplificadas en un Stratagene Mx3000P en las condiciones siguientes: 95°C durante 10 minutos, seguido de 45 ciclos de 90ºC durante 30 segundos y 60ºC durante 1 minuto.
Las casetes de ADN que albergan mutaciones puntuales que se emplean como controles positivos fueron construidas en base a un método descrito por Higuchi et al.[2]. Resumidamente, se usaron cebadores exteriores y mutámeros correspondientes para generar medias casetes con extremos complementarios, conteniendo cada media casete una base mutante. Dichos productos de PCR fueron mezclados y amplificados con cebadores anidados interiores. El auto-cebado de las medias casetes complementarias y la amplificación posterior crearon un producto final con una base mutada. Los productos fueron secuenciados para asegurar que se había creado la secuencia correcta. Este proceso se repitió para cada mutación de interés. La casete de ADN se mezcló con una cantidad igual de ADN genómico para crear un control 100% positivo.
Ejemplo 1
Para determinar la especificidad de las reacciones y los cebadores se llevó a cabo cada ensayo con 5-50 ng de ADN genómico por reacción para determinar la señal de rotura producida por la extensión de cebador no coincidente. Para cada reacción se definió un valor ΔCt (Ct de control -Ct de mutación) (Ct = ciclo umbral, del inglés "threshold cycle"). Las reacciones se llevaron a cabo seis veces para cada concentración de ADN y se repitieron por triplicado en momentos diferentes para definir un valor de ΔCt de corte por debajo del cual se puede decir que cualquier amplificación es debida a la presencia de secuencia mutante y que no es debida a una señal de rotura. Se determinó que el valor de ΔCt de corte era 1 Ct inferior al menor valor de ΔCt observado en todas las reacciones de cada ensayo. Para los ensayos de H1047R y H1047L, se definió el corte de ΔCt como 12, para el ensayo de E542K el valor de corte de ΔCt fue de 9 y para el ensayo de E545K el corte de ΔCt fue 8.
Ejemplo 2
Para determinar la sensibilidad del ensayo, se diluyeron 5 copias de ADN mutante en concentraciones variables de ADN genómico para producir concentraciones finales de 5, 2, 1, 0,5 y 0,1 % de ADN mutante respecto al natural. La Tabla 1 ilustra la sensibilidad de los 4 ensayos ARMS. La tabla muestra los valores de ΔCt para concentraciones
E10158519
05-03-2015
El ensayo ARMS identificó significativamente más mutaciones en las muestras clínicas que las observadas mediante secuenciamiento directo. Las mezclas de línea celular confirman que este ensayo es más sensible que el secuenciamiento para detectar las mutaciones PI3KCA de interés. Es probable que la heterogeneidad de las muestras clínicas que contienen tanto tejido normal como tumoral signifique que en algunos casos la incidencia de la
5 mutación esté por debajo de la detectable por métodos de secuenciamiento, y como tal el ensayo ARMS es más adecuado para una aplicación clínica. La desventaja es que solo se detectan determinadas mutaciones específicas de ARMS. Sin embargo, en esta serie de 279 muestras solo se detectó una única mutación en el exón 9 ó 20 del gen PI3KCA para cuya detección el ensayo ARMS no estaba diseñado.
En resumen, los ejemplos demuestran que la presente invención proporciona un ensayo sensible de alta capacidad
10 para la detección de las 4 mutaciones más comunes del gen PIK3CA. Dicho ensayo puede aplicarse a cantidades pequeñas de ADN y puede detectar niveles bajos de PIK3CA mutante en una muestra.
E10158519
05-03-2015
Referencias:
1. Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science 2004; 304(5670): 554.
2. Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA 5 fragments: study of protein and DNA interactions. Nucleic Acids Res 1988; 16(15): 7351-67.
- 3.
- Wu G, Xing M, Mambo E, et al. Somatic mutation and gain of copy number of PIK3CA in human breast cancer. Breast Cancer Res 2005; 7(5): R609-16.
- 4.
- Levine DA, Bogomolniy F, Yee CJ, et al. Frequent mutation of the PIK3CA gene in ovarian and breast cancers. Clin Cancer Res 2005; 11(8):2875-8.
10 5. Velho S, Oliveira C, Ferreira A, et al. The prevalence of PIK3CA mutations In gastric and colon cancer. Eur J Cancer 2005;41 (11): 1649-54.
6. Lee JW, Soung YH, Kim SY, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 2005; 24(8): 1477-80.
7. Bachman KE, Argani P, Samuels Y, et al. The PIK3CA gene is mutated with high frequency in human breast 15 cancers. Cancer Biol Ther 2004; 3(8): 772-5.
- 8.
- Campbell IG, Russell SE, Choong DY, et al. Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004; 64(21): 7678-81.
- 9.
- Omholt K, Krockel D, Ringborg U, Hansson J. Mutations of PIK3CA are rare in cutaneous melanoma. Melanoma Res 2006;16(2):197-200,
20 10. Wang Y, Helland A, Holm R, Kristensen GB, Borresen-Dale AL. PIK3CA mutations In advanced ovarian carcinomas. Hum Mutat 2005;25(3):322.
Claims (1)
-
imagen1
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB0719034 | 2007-09-28 | ||
| GB0719034A GB2453173A (en) | 2007-09-28 | 2007-09-28 | Polynucleotide primers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2532141T3 true ES2532141T3 (es) | 2015-03-24 |
Family
ID=38701889
Family Applications (6)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES10158523.0T Active ES2531994T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
| ES12169604T Active ES2531055T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
| ES10158519.8T Active ES2532141T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
| ES12169599.3T Active ES2620289T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
| ES12169586.0T Active ES2622885T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
| ES12169594.4T Active ES2532225T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES10158523.0T Active ES2531994T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
| ES12169604T Active ES2531055T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
Family Applications After (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES12169599.3T Active ES2620289T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
| ES12169586.0T Active ES2622885T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores polinucleótidos para detectar mutaciones PIK3CA |
| ES12169594.4T Active ES2532225T3 (es) | 2007-09-28 | 2008-09-29 | Cebadores de polinucleótidos para detectar mutaciones de PIK3CA |
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| Country | Link |
|---|---|
| US (4) | US8901285B2 (es) |
| EP (7) | EP2505671B1 (es) |
| JP (1) | JP5719593B2 (es) |
| KR (4) | KR101548758B1 (es) |
| CN (4) | CN103834724B (es) |
| AU (1) | AU2008303400B2 (es) |
| BR (1) | BRPI0817444A2 (es) |
| CA (2) | CA3010064A1 (es) |
| ES (6) | ES2531994T3 (es) |
| GB (1) | GB2453173A (es) |
| MX (1) | MX2010003486A (es) |
| RU (1) | RU2491289C2 (es) |
| WO (1) | WO2009040557A2 (es) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10190171B2 (en) | 2007-09-28 | 2019-01-29 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3246332B1 (en) * | 2010-01-12 | 2019-07-10 | Siemens Healthcare Diagnostics Inc. | Oligonucleotides and methods for detecting pik3ca mutations |
| JP2013081450A (ja) * | 2011-09-27 | 2013-05-09 | Arkray Inc | 多型検出用プローブ、多型検出方法、薬効判定方法及び多型検出用試薬キット |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10190171B2 (en) | 2007-09-28 | 2019-01-29 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
| US10196692B2 (en) | 2007-09-28 | 2019-02-05 | Qiagen Manchester Limited | Polynucleotide primers for detecting PIK3CA mutations |
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