WO2006111586B1 - Method for the in vitro determination of the degree of methylation of the line-1 promoter - Google Patents

Method for the in vitro determination of the degree of methylation of the line-1 promoter

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Publication number
WO2006111586B1
WO2006111586B1 PCT/ES2005/000205 ES2005000205W WO2006111586B1 WO 2006111586 B1 WO2006111586 B1 WO 2006111586B1 ES 2005000205 W ES2005000205 W ES 2005000205W WO 2006111586 B1 WO2006111586 B1 WO 2006111586B1
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WO
WIPO (PCT)
Prior art keywords
methylation
line
degree
seq
promoter
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Application number
PCT/ES2005/000205
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Spanish (es)
French (fr)
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WO2006111586A3 (en
WO2006111586A2 (en
Inventor
Ena Xabier Aguirre
Gomez Jose Roman
Velasco Antonio Jimenez
Cardoso Felipe Prosper
Original Assignee
Proyecto Biomedicina Cima Sl
Medica E Investigacion A M I A
Fundacion Imabis
Ena Xabier Aguirre
Gomez Jose Roman
Velasco Antonio Jimenez
Cardoso Felipe Prosper
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Proyecto Biomedicina Cima Sl, Medica E Investigacion A M I A, Fundacion Imabis, Ena Xabier Aguirre, Gomez Jose Roman, Velasco Antonio Jimenez, Cardoso Felipe Prosper filed Critical Proyecto Biomedicina Cima Sl
Priority to PCT/ES2005/000205 priority Critical patent/WO2006111586A2/en
Publication of WO2006111586A2 publication Critical patent/WO2006111586A2/en
Publication of WO2006111586A3 publication Critical patent/WO2006111586A3/en
Publication of WO2006111586B1 publication Critical patent/WO2006111586B1/en

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    • 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
    • C12Q1/6876—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a method for the in vitro determination of the degree of methylation of the LINE-1 promoter, which is characterised in that it comprises genomic DNA amplification with specific amplification primers.

Claims

24REIVINDICACIONES MODIFICADAS[recibidas por Ia Oficina Internacional el 18 de agosto de 2006 (18.08.06)]MODIFICADO SEGÚN EL ARTICULO 19(1): REIVINDICACIONES 24 MODIFIED REVINDICATIONS [received by the International Bureau on August 18, 2006 (08-18.06)] MODIFIED ACCORDING TO ARTICLE 19 (1): CLAIMS
1. Procedimiento para la determinación in vi tro del grado de metilación del promotor de LINE-I, caracterizado porque comprende: a) amplificar una muestra de ADN genómico mediante un par de cebadores de amplificación específicos de secuencias no metiladas y un par de cebadores de amplificación específicos de secuencias metiladas, donde ambos pares de cebadores :A method for the determination of the degree of methylation of the LINE-I promoter, characterized in that it comprises: a) amplifying a sample of genomic DNA by means of a pair of specific amplification primers of non-methylated sequences and a pair of primers of Specific amplification of methylated sequences, where both pairs of primers:
-poseen un tamaño comprendido entre 10 y 30 nucleótidos,-have a size between 10 and 30 nucleotides,
-hibridan selectivamente con una secuencia comprendida entre los nucleótidos 49 y 420 de la secuencia consenso del promotor de LINE-I,selectively inhibit with a sequence between nucleotides 49 and 420 of the consensus sequence of the LINE-I promoter,
-al menos uno de ellos hibrida selectivamente con una secuencia comprendida entre los nucleótidos 100 y 150, o entre los nucleótidos 240 y 290 de dicha secuencia consenso del promotor; y b) cuantificar el grado de metilación de la secuencia analizada .- at least one of them hybridizes selectively with a sequence comprised between nucleotides 100 and 150, or between nucleotides 240 and 290 of said consensus sequence of the promoter; and b) quantifying the degree of methylation of the sequence analyzed.
2. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según la reivindicación 1, caracterizado porque dichos cebadores poseen un tamaño de entre 15 y 25 nucleótidos .2. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to claim 1, characterized in that said primers have a size between 15 and 25 nucleotides.
3. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 1 o 2, caracterizado porque3. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to one of claims 1 or 2, characterized in that
HOJA MODIFICADA (ARTÍCULO 19) al menos uno de los cebadores de amplificación está seleccionado entre: SEQ. ID. NO: 1, SEQ. ID. NO : 2 , SEQ. ID. NO: 3, y SEQ. ID. NO : 4.MODIFIED LEAF (ARTICLE 19) at least one of the amplification primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, and SEQ. ID. NO: 4
4. Procedimiento para la determinación in vi tro del grado de metilación del promotor de LINE-I según la reivindicación 1, caracterizado porque dichos cebadores específicos para secuencias metiladas poseen las secuencias correspondientes a SEQ. ID. NO: 1 y 2. 4. Procedure for the determination of the degree of methylation of the promoter of LINE-I according to claim 1, characterized in that said primers specific for methylated sequences possess the sequences corresponding to SEQ. ID. NO: 1 and 2.
5. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según la reivindicación 1, caracterizado porque dichos cebadores específicos para secuencias no metiladas poseen secuencias correspondientes a SEQ. ID. NO: 3 y 4.5. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to claim 1, characterized in that said primers specific for non-methylated sequences possess sequences corresponding to SEQ. ID. NO: 3 and 4
6. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 1 a 5, caracterizado porque dicho procedimiento es una PCR sensible a la metilación.6. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to one of claims 1 to 5, characterized in that said method is a PCR sensitive to methylation.
7. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 1 a 6, caracterizado porque dicha PCR sensible a la metilación es de cuantificación en tiempo real.7. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to one of claims 1 to 6, characterized in that said methylation-sensitive PCR is of real time quantification.
8. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según la reivindicación 1, caracterizado porque un grado de metilación inferior al 80% se relaciona con presencia de leucemia. 268. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to claim 1, characterized in that a methylation degree lower than 80% is related to the presence of leukemia. 26
9. Procedimiento para la determinación in vi tro del grado de metilación del promotor de LINE-I según la reivindicación 8, caracterizado porque dicho grado de metilación es inferior al 70%. 9. Procedure for the determination of the degree of methylation of the LINE-I promoter according to claim 8, characterized in that said degree of methylation is less than 70%.
10. Procedimiento para la determinación in vitro del 'grado de metilación del promotor de LIME-I según la reivindicación 8, caracterizado porque dicho grado de metilación es inferior al 50%.10. Procedure for the in vitro determination of the degree of methylation of the LIME-I promoter according to claim 8, characterized in that said degree of methylation is less than 50%.
11. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 8 a 10, caracterizado porque dicha leucemia es una leucemia aguda o una leucemia crónica .11. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to one of claims 8 to 10, characterized in that said leukemia is an acute leukemia or a chronic leukemia.
12. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según la reivindicación 11, caracterizado porque dicha leucemia está seleccionada entre: una leucemia mieloide crónica, una leucemia mieloide aguda, una leucemia linfocítica aguda o una leucemia linfocítica crónica.12. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to claim 11, characterized in that said leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia or chronic lymphocytic leukemia .
13. Procedimiento para la determinación in vitro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 11 o 12, caracterizado porque dicha determinación permite determinar la evolución de dicha leucemia.13. Procedure for the in vitro determination of the methylation degree of the LINE-I promoter according to one of claims 11 or 12, characterized in that said determination makes it possible to determine the evolution of said leukemia.
14. Procedimiento para la determinación in vi tro del grado de metilación del promotor de LINE-I según una de las reivindicaciones 11 o 12, caracterizado porque permite determinar la evolución de dicha leucemia en respuesta a tratamientos. 2714. Procedure for the determination of the degree of methylation of the promoter of LINE-I according to one of claims 11 or 12, characterized in that it allows determining the evolution of said leukemia in response to treatments. 27
15. Un cebador para la amplificación del promotor de LINE-I según el procedimiento descrito en una de las reivindicaciones 1 a 14, caracterizado porque su secuencia está seleccionada entre: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 y SEQ. ID. NO: 4.15. A primer for the amplification of the LINE-I promoter according to the method described in one of claims 1 to 14, characterized in that its sequence is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4
16. Kit para determinar el grado de metilación del promotor de LINE-I, caracterizado porque comprende un par de cebadores de amplificación específicos de secuencias no metiladas y un par de cebadores de amplificación específicos de secuencias metiladas.16. Kit for determining the methylation degree of the LINE-I promoter, characterized in that it comprises a pair of amplification primers specific for non-methylated sequences and a pair of amplification primers specific for methylated sequences.
17. Kit para determinar el grado de metilación del promotor de LINE-I según la reivindicación 16, caracterizado porque ambos pares de cebadores :17. Kit for determining the methylation degree of the LINE-I promoter according to claim 16, characterized in that both pairs of primers:
-poseen un tamaño comprendido entre 10 y 30 nucleótidos,-have a size between 10 and 30 nucleotides,
-hibridan selectivamente con una secuencia comprendida entre los nucleótidos 49 y 420 de la secuencia consenso del promotor de LINE-I, -al menos uno de ellos híbrida selectivamente con una secuencia comprendida entre los nucleótidos 100 y 150, o entre los nucleótidos 240 y 290 de dicha secuencia consenso del promotor .selectively inhibit with a sequence comprised between nucleotides 49 and 420 of the consensus sequence of the LINE-I promoter, at least one of them selectively hybridizes with a sequence comprised between nucleotides 100 and 150, or between nucleotides 240 and 290 of said consensus sequence of the promoter.
18. Kit para determinar el grado de metilación del promotor de LINE-I según una de las reivindicaciones18. Kit for determining the methylation degree of the LINE-I promoter according to one of the claims
16 o 17, caracterizado porque al menos uno de los cebadores está seleccionado entre: SEQ. ID. NO :1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 y SEQ. ID. NO : 4.16 or 17, characterized in that at least one of the primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4
19. Kit para determinar el grado de metilación del promotor de LINE-I según una de las reivindicaciones19. Kit for determining the methylation degree of the LINE-I promoter according to one of the claims
HQJA MODIFICADA (ARTICULO 19) 28MODIFIED HQJA (ARTICLE 19) 28
16 a 18, caracterizado porque el par de cebadores de amplificación específico de secuencias metiladas son SEQ. ID. NO: 1 y 2, y el par de cebadores de amplificación específico de secuencias no metiladas son SEQ. ID. NO: 3 y 4.16 to 18, characterized in that the pair of specific amplification primers of methylated sequences are SEQ. ID. NO: 1 and 2, and the pair of specific amplification primers of non-methylated sequences are SEQ. ID. NO: 3 and 4
20. Kit para determinar el grado de metilación del promotor de LINE-I según una de las reivindicaciones 16 a 19, caracterizado porque comprende reactivos apropiados para realizar un método de PCR sensible a la metilación.20. Kit for determining the methylation degree of the LINE-I promoter according to one of claims 16 to 19, characterized in that it comprises appropriate reagents for carrying out a methylation-sensitive PCR method.
HQJA MODIFICADA (ARTICULO 19) MODIFIED HQJA (ARTICLE 19)
PCT/ES2005/000205 2005-04-20 2005-04-20 Method for the in vitro determination of the degree of methylation of the line-1 promoter WO2006111586A2 (en)

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PCT/ES2005/000205 WO2006111586A2 (en) 2005-04-20 2005-04-20 Method for the in vitro determination of the degree of methylation of the line-1 promoter

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WO2006111586A2 WO2006111586A2 (en) 2006-10-26
WO2006111586A3 WO2006111586A3 (en) 2006-12-14
WO2006111586B1 true WO2006111586B1 (en) 2007-01-11

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2210954T3 (en) * 2009-01-22 2011-10-10 Univ Duesseldorf H Heine Determination of DNA methylation level
CN114182005A (en) * 2021-10-29 2022-03-15 上海普然生物科技有限公司 Detection kit for LINE-1 methylation detection and detection method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10032529A1 (en) * 2000-06-30 2002-02-07 Epigenomics Ag Diagnosis of major genetic parameters within the Major Histocompatibility Complex (MHC)
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
EP1370685A2 (en) * 2000-04-06 2003-12-17 Epigenomics AG Diagnosis of diseases associated with dna repair
AU2001289617A1 (en) * 2000-06-30 2002-01-08 Epigenomics Ag Diagnosis of diseases associated with signal transduction
DE10061338A1 (en) * 2000-12-06 2002-06-20 Epigenomics Ag Diagnosis of diseases associated with angiogenesis

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WO2006111586A2 (en) 2006-10-26

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