WO2009068211A1 - Procédé pour déterminer la présence ou l'absence de plusieurs séquences cibles dans un échantillon biologique - Google Patents

Procédé pour déterminer la présence ou l'absence de plusieurs séquences cibles dans un échantillon biologique Download PDF

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Publication number
WO2009068211A1
WO2009068211A1 PCT/EP2008/009782 EP2008009782W WO2009068211A1 WO 2009068211 A1 WO2009068211 A1 WO 2009068211A1 EP 2008009782 W EP2008009782 W EP 2008009782W WO 2009068211 A1 WO2009068211 A1 WO 2009068211A1
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WIPO (PCT)
Prior art keywords
amplification reaction
base pairs
primer pairs
determined
pairs
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PCT/EP2008/009782
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German (de)
English (en)
Inventor
Wolfgang Mann
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Olympus Life Science Research Europa Gmbh
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Publication of WO2009068211A1 publication Critical patent/WO2009068211A1/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for the simultaneous determination of the presence and / or absence of at least two different target sequences in a biological sample by means of at least one amplification reaction in which at least 3 primer pairs are used for each target sequence to be determined, and subsequent detection of the in the at least one amplification reaction obtained amplificates by an electrophoresis method.
  • Method for determining the presence and / or absence of target sequences in a biological sample i.
  • the detection of the presence or absence of a predetermined nucleic acid sequence, such as a particular chromosome, gene or gene segment are used in many technical fields. Only for example applications in medical diagnostics, in forensics or in genetic engineering are mentioned.
  • a PCR (“polymerase chain reaction” or “polymerase chain reaction”) is usually carried out in which at least one primer pair is used, which is adapted thereto in that in the PCR, a nucleic acid sequence present in the target sequence is amplified with a predetermined length.
  • the PCR product can also be further characterized, for example by sequencing.
  • the conclusion that the target sequence is not contained in the genome of the single cell would be associated with a high risk of error.
  • the validity of the result obtained at the single cell can be significantly increased. For example, if the probability of amplification of a sequence from the target sequence in the single-cell PCR per primer pair is 50%, then the likelihood of not obtaining a single amplification product in a PCR with 5 different primer pairs, although the corresponding target sequences in the genome the single cell, at about 3%. If, however, 10 or even 100 different primer pairs used, the probability at about 0, 1% and 10- 29%.
  • 10 different PCRs can be carried out for the abovementioned examination with, for example, 10 different primer pairs, wherein in each case one primer pair is used per PCR.
  • 10 different reaction mixtures must be handled and analyzed after the PCR, which is very time consuming and costly. For this reason, in this case often only a (multiplex) PCR is performed, in which the 10 different primer pairs are used.
  • the various PCR products are present in a sample, so that they can be characterized in parallel, for example by gel electrophoresis.
  • the different primer pairs are in practice selected such that each primer pair yields a PCR product of a certain length but different from that of any other PCR product.
  • target sequences are often simultaneously characterized in a biological sample, ie, for example, it is determined in parallel whether an individual cell, such as a polar body, has three specific chromosomes or not.
  • an individual cell such as a polar body
  • more pairs of primers are to be used in the multiplexex PCR, and accordingly more PCR PCR amplifiers are required after the PCR has been carried out.
  • the object of the present invention is therefore to provide a method for the simultaneous determination of the presence and / or absence of at least two different target sequences in a biological sample by means of an amplification reaction in which several different primer pairs are used per target sequence to be determined, which even when using a single cell can be done easily, quickly and inexpensively.
  • this object is achieved by a method for determining the presence and / or absence of at least two different target sequences in a biological sample comprising:
  • the at least one amplification reaction is a PCR or an LCR in which at least 3 different primer pairs are used per target sequence to be determined, and then ii) the detection of the amplicons obtained in the at least one amplification reaction comprising the implementation an electrophoresis method,
  • primer pairs for each of the at least two target sequences to be determined are selected such that all of the amplificates obtainable in the amplification reaction per target sequence to be determined with the primer pairs differ in their length by a maximum of x base pairs and in that
  • all amplicons obtainable from the amplification reaction per target sequence to be determined with the primer pairs differ in their length from the amplicons obtainable with the primer pairs for each other target sequence by at least y base pairs, where x ⁇ z and y> z, z is the resolving power of the electrophoresis method performed in step ii).
  • the term resolving power of the electrophoresis method carried out in method step ii) is understood to mean the number of base pairs or bases around which at least two different nucleic acid fragments must differ, so that these two nucleic acid fragments are separated from one another in the specifically performed electrophoresis method in that these are present as distinguishable bands after completion of the electrophoresis.
  • the resolution unit is base pairs or bases, depending on whether double-stranded nucleic acid is electrophoretically separated in a non-denaturing gel (base pairs) or whether single-stranded nucleic acid is electrophoresed in a non-denaturing gel or nucleic acid in a denaturing gel (bases).
  • the resolution of an electrophoresis process depends on many factors, in particular the type of gel used. For example, the resolution of polyacrylamide gel electrophoresis is greater than that of agarose gel electrophoresis. Furthermore, the resolving power also depends significantly on the (crosslinking) The greater the (crosslinking) density of the gel, the greater the resolution. Apart from that, the resolving power also depends on the concrete process parameters, such as the voltage applied in the electrophoresis and in particular the duration of the electrophoresis. The longer the duration of the electrophoresis, ie the time for which tension is applied to the gel, the greater the resolution.
  • the resolution z is determined by the sum of all of the aforementioned parameters, which are set in the electrophoresis method actually used in the method according to the invention.
  • Polyacrylamide gel electrophoresis up to 1 b or bp
  • Capillary gel electrophoresis up to 1 b or bp.
  • a method for determining the presence and / or absence of a target sequence in the context of the present invention comprises both Determining whether or not the target sequence is present, as well as determining the number of target sequences present, whether monosomy, disomy, or trisomy.
  • the primer pairs for each of the at least two target sequences to be determined are selected so that, firstly, all of the amplificates obtainable in the amplification reaction per target sequence to be determined with the primer pairs are maximally x base pairs in length, ie less than the resolution z of the used in the process step ii)
  • Electrophoresis method are distinguished, are obtained for each primer pairs used for each target sequence to be determined amplificates with indistinguishable by the electrophoresis method length.
  • the primer pairs are selected for each of the at least two target sequences to be determined such that, secondly, all of the amplicons obtainable with the primer pairs in the amplification reaction per amplification reaction are different in length from those in the amplification reaction for any other target sequence amplicons available with the primer pairs differ by at least y base pairs, ie by more than the resolution z of the electrophoresis method used in method step ii), all amplificates to be determined with the primer pairs obtained in the electrophoresis method used are from those for each distinguishable from other target sequences.
  • the inventive method is much less labor, time and cost intensive than the known from the prior art method.
  • the presence of the target chromosome can be concluded with almost 100% certainty if at least one PCR product for a primer pair which is specific for a sequence segment in the target chromosome is obtained in the PCR.
  • All primer pairs for chromosome 13 are designed to include all of these primer pairs the amplification yield a PCR product with a length of 100 base pairs.
  • all primer pairs for chromosome 18 are designed so that all of these primer pairs will yield a 300 base pair PCR product upon amplification, whereas all primer pairs for chromosome 21 will be designed so that all of these primer pairs will be amplified give a PCR product with a length of 500 base pairs.
  • the PCR batch is applied to an agarose gel and electrophoretically separated before the agarose gel is stained with ethidium bromide and the number of bands obtained is determined.
  • a maximum of three different DNA bands can be obtained in the agarose gel, namely bands at 100, 300 and 500 base pairs when chromosomes 13, 18 and 21 are present in the polar body.
  • the requirement for the separation efficiency of the gel is very low.
  • only the presence or absence of PCR products with three specific lengths has to be determined, so that the process can be carried out very quickly and with little work.
  • target sequence is understood to mean any nucleic acid sequence
  • a biological sample is understood as meaning a sample in which nucleic acid from at least one individual is contained.
  • the biological sample may also contain nucleic acid from two or more individuals, so be a mixed sample.
  • the primer pairs are selected for each of the at least two target sequences to be characterized in such a way that all of the amplificates obtainable with the primer pairs in the amplification reaction have essentially the same length, namely a maximum of x base pairs in length where x is smaller than the resolution of the electrophoresis method used, ie all of the Amplif ⁇ kate available in the Amplif ⁇ kationsreak- tion per target sequence to be determined with the primer pairs with the electrophoresis method used in terms of their length are indistinguishable.
  • the amplificates for a target sequence to differ in length by as much as the resolution of the electrophoresis technique, some freedom in the design of the primers is gained.
  • the individual amplifcates obtained for each target sequence have essentially the same length, so that, when electrophoresis is carried out appropriately, a sharp DNA band is still obtained.
  • step ii) of the process according to the invention it is possible to use all electrophoresis processes known to the person skilled in the art.
  • electrophoresis processes known to the person skilled in the art.
  • agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis may be mentioned.
  • the type of method used to determine the length of the amplified cells obtained in the at least one amplification reaction can be selected as a function of the difference in length between amplifications of different target sequences and in dependence on the slight difference in the length of the individual amplicons obtained per target sequence.
  • an agarose gel for example, may be used for this purpose, whereas otherwise a high-resolution polyacrylamide gel should be used.
  • the primer pairs for all of the at least two target sequences to be determined are selected so that all of the target sequence to be determined in the amplification reaction per target sequence pairable Amplif ⁇ kate available in their length by a maximum of 10 base pairs, more preferably by a maximum of 5 base pairs, further preferably by a maximum of 4 base pairs, more preferably by a maximum of 3 base pairs, more preferably by a maximum of 2 base pairs and most preferably distinguished by a maximum of 1 base pair.
  • the primer pairs for each of the at least two target sequences to be characterized are selected so that all of the amplifcates obtainable with the primer pairs for each amplification reaction are identical in length, ie differ in their length by 0 base pairs.
  • the primer pairs for each of the at least two target sequences to be determined are selected such that all of the amplicons obtainable from the amplification reaction per target sequence to be determined with the primer pairs are identical in length to the amplicons obtainable from the amplification reaction for each other target sequence with the primer pairs differ by at least 25 base pairs, more preferably by at least 50 base pairs, and most preferably by at least 100 base pairs.
  • the method comprises the simultaneous determination of the presence and / or absence of at least two different NEN target sequences in a biological sample, the process step of performing at least one amplification reaction, ie a PCR, an LCR, multiple PCR's or multiple LCR's.
  • a (multiplex) PCR or a (multiplex) LCR is carried out, in which all the primer pairs required for determining the presence and / or absence of the at least two different target sequences are used, if the number of primers used primer pairs a multiplex PCR or a (multi-lex) LCR allow.
  • the number of primers used is too large for a single multiplex PCR or (multiplex) LCR
  • the individual samples can then be examined individually with respect to the existing PCR products or LCR products or, preferably, first mixed together before the length of the resulting PCR products or LCR products is determined.
  • This embodiment can be used, for example, when target sequences from different individuals are to be characterized, wherein initially with the nucleic acid of each individual a PCR or LCR is performed before the individual PCR approaches or LCR approaches are mixed together and then the length the PCR products or LCR products obtained is determined.
  • the method according to the invention is particularly suitable for determining the presence and / or absence of at least two different target sequences in a biological sample which contains only a small amount of nucleic acid. Therefore, the method according to the invention is particularly suitable for determining the presence and / or absence of at least two different target sequences in a single cell or in a polar body.
  • the at least two target sequences to be characterized may each be any nucleic acid sequence.
  • the at least two target sequences to be determined may each independently, be a chromosome, a gene, a gene segment or a non-coding nucleic acid sequence.
  • three different target sequences can be characterized for their presence or absence, each of which is a chromosome.
  • the method according to the invention is particularly suitable for the use of many pairs of primers, it is proposed in the invention that 3 to 50 different target sequences, preferably 5 to 25 different target sequences and particularly preferably 7 to 15 different target sequences with regard to their presence or absence be added determine.
  • the number of primer pairs to be used per target sequence to be determined depends on the one hand on the desired statistical certainty of the result and on the other hand on the amount of nucleic acid contained in the biological sample.
  • the larger the amount of nucleic acid used the fewer primer pairs per target sequence to be characterized suffice to obtain a meaningful result.
  • the more primer pairs must be used the greater the statistical reliability of the result should be.
  • in the amplification reaction per target sequence to be determined preferably 3 to 50 primer pairs, more preferably 4 to 25 primer pairs, are very particular preferably 5 to 20 primer pairs and most preferably, in particular in the case of the use of a single cell as a biological sample, 10 to 15 primer pairs used.
  • the at least 3 primer pairs to be used for each target sequence to be determined are adapted to amplify non-overlapping regions of the target sequence to be characterized in an amplification reaction.
  • the at least 3 primer pairs to be used for each target sequence to be determined may have binding sites in the coding region of the target sequence or in the non-coding region of the target sequence.
  • the present invention relates to a method for the simultaneous determination of the presence and / or absence of at least three different target sequences in a single cell comprising:
  • the at least one amplification reaction is a PCR or an LCR in which at least 3 different primer pairs are used per target sequence to be determined, and then ii) the detection of the amplicons obtained in the at least one amplification reaction comprising the implementation an electrophoresis method,
  • primer pairs for each of the at least three target sequences to be determined are selected such that all of the amplificates obtainable in the amplification reaction per target sequence to be determined with the primer pairs have the same length and
  • all amplicons obtainable from the amplification reaction per target sequence to be determined with the primer pairs differ in their length from the amplicons obtainable in the amplification reaction for each other target sequence with the primer pairs.
  • the primer pairs for each of the at least three target sequences to be determined are selected such that all of the amplicons obtainable with the primer pairs in the amplification reaction for each amplification reaction are at least equal in length to the amplicons obtainable from the amplification reaction for each other target sequence with the primer pairs 1 base pair, preferably by at least 3 base pairs, more preferably by at least 5 base pairs, and most preferably by at least 10 base pairs.
  • the primer pairs for each of the at least two target sequences to be determined are preferably selected so that all of the amplicons obtainable in the amplification reaction per target sequence to be determined with the primer pairs are of the same length as in the Amplification reaction for each other target sequence with the primer pairs available amplificates differ by at least 25 base pairs, more preferably by at least 50 base pairs and most preferably by at least 100 base pairs.
  • This method is particularly suitable for determining the presence and / or absence of all 46 chromosomes in a diploid human body. single cell or all 23 chromosomes in a haploid human single cell.
  • a further subject of the present invention is a kit for the simultaneous determination of the presence and / or absence of at least two different target sequences in a biological sample by means of at least one amplification reaction, comprising:
  • a protocol for carrying out a PCR or an LCR as amplification reaction ii) a protocol for carrying out an electrophoresis method, wherein the resolving power of the electrophoresis method described in the protocol is z base pairs or bases, and iii) in each case at least three mutually different primer pairs for each of the at least two different target sequences, wherein
  • the primer pairs for each of the at least two target sequences to be determined are selected such that
  • all of the amplicons obtainable from the amplification reaction per target sequence to be determined with the primer pairs differ by at least y base pairs from the amplicons obtainable with the primer pairs for each other target sequence in the amplification reaction, where x ⁇ z and y> z ,
  • the primer pairs contained in the kit according to the invention are selected in this way in that the resulting difference in length x of the amplificates is less than or equal to 10 base pairs, more preferably less than or equal to 4 base pairs, more preferably less than or equal to 3 base pairs, more preferably less than or equal to 2 base pairs, most preferably less than or equal to 1 base pair most preferably 0 base pairs.
  • the primer pairs contained in the kit according to the invention are selected such that the resulting length difference y is at least 10 base pairs, more preferably at least 25 base pairs, most preferably at least 50 base pairs, and most preferably at least 100 base pairs.
  • the kit according to the invention contains so many and so selected primer pairs that it is suitable for determining the presence or absence of all human chromosomes present in the biological sample, i. of 23 or 46 human chromosomes.

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  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne un procédé pour déterminer la présence ou l'absence d'au moins deux séquences cibles différentes dans un échantillon biologique, ce procédé consistant à i) effectuer au moins une réaction d'amplification qui est une amplification PCR ou LCR, dans laquelle au moins trois paires d'amorces différentes sont utilisées par séquence cible à déterminer, puis à ii) mettre en évidence les produits d'amplification obtenus lors de la ou des amplification(s) par un procédé d'électrophorèse. Les paires d'amorces pour chacune des au moins deux séquences cibles à déterminer sont sélectionnées de manière à ce que tous les produits d'amplification obtenus au moyen des paires d'amorces par séquence cible à déterminer lors de l'amplification présentent une longueur qui se différencie par x paires de base au maximum et par au moins y paires de base des produits d'amplification obtenus au moyen des paires d'amorces pour chaque autre séquence cible lors de l'amplification, x étant inférieur ou égal à z et y étant supérieur à z, z représentant le pouvoir séparateur du procédé d'électrophorèse effectué à l'étape (ii).
PCT/EP2008/009782 2007-11-30 2008-11-19 Procédé pour déterminer la présence ou l'absence de plusieurs séquences cibles dans un échantillon biologique WO2009068211A1 (fr)

Applications Claiming Priority (2)

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DE102007057698.8 2007-11-30
DE200710057698 DE102007057698A1 (de) 2007-11-30 2007-11-30 Verfahren zur Bestimmung der An- und/oder Abwesenheit von mehreren Zielsequenzen in einer biologischen Probe

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DE102015111329B4 (de) * 2015-07-13 2017-02-02 Bernd-Peter Ernst Verfahren zum Bestimmen einer relativen Häufigkeit von verschiedenen Genen oder Chromosomen eines Genoms in einer Probe

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031646A1 (fr) * 2001-10-12 2003-04-17 The University Of Queensland Selection et amplification de marqueurs genetiques multiples
US20060057605A1 (en) * 2004-03-22 2006-03-16 Isis Pharmaceuticals, Inc. Compositions for use in identification of viral hemorrhagic fever viruses
WO2006094360A1 (fr) * 2005-03-11 2006-09-14 Molecular Plant Breeding Nominees Ltd Procede destine a amplifier les acides nucleiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031646A1 (fr) * 2001-10-12 2003-04-17 The University Of Queensland Selection et amplification de marqueurs genetiques multiples
US20060057605A1 (en) * 2004-03-22 2006-03-16 Isis Pharmaceuticals, Inc. Compositions for use in identification of viral hemorrhagic fever viruses
WO2006094360A1 (fr) * 2005-03-11 2006-09-14 Molecular Plant Breeding Nominees Ltd Procede destine a amplifier les acides nucleiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FINDLAY I ET AL: "PREIMPLANTATION GENETIC DIAGNOSIS USING FLUORESCENT POLYMERASE CHAIN REACTION: RESULTS AND FUTURE DEVELOPMENTS", JOURNAL OF ASSISTED REPRODUCTION AND GENETICS, PLENUM PUBLISHING, US, vol. 16, no. 4, 1 January 1999 (1999-01-01), pages 199 - 206, XP008033799, ISSN: 1058-0468 *
FINDLAY I ET AL: "USING MF-PCR TO DIAGNOSE MULTIPLE DEFECTS FROM SINGLE CELLS: IMPLICATIONS FOR PGD", MOLECULAR AND CELLULAR ENDOCRINOLOGY, ELSEVIER IRELAND LTD, IE, vol. 183, 1 January 2001 (2001-01-01), pages S05 - S12, XP001199454, ISSN: 0303-7207 *
LINDQVIST A-K B ET AL: "CHROMOSOME-SPECIFIC PANELS OF TRI- AND TETRANUCLEOTIDE MICROSATELLITE MARKERS FOR MULTIPLEX FLUORESCENT DETECTION AND AUTOMATED GENOTYPING: EVALUATION OF THEIR UTILITY IN PATHOLOGY AND FORENSICS", PCR METHODS & APPLICATIONS, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 6, no. 12, 1 January 1996 (1996-01-01), pages 1170 - 1176, XP009005321, ISSN: 1054-9803 *
MCARTHUR ET AL: "Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts", FERTILITY AND STERILITY, ELSEVIER SCIENCE INC, NEW YORK, NY, US, vol. 84, no. 6, 1 December 2005 (2005-12-01), pages 1628 - 1636, XP022093808, ISSN: 0015-0282 *

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