EP1960537A1 - Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus - Google Patents

Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus

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Publication number
EP1960537A1
EP1960537A1 EP06818281A EP06818281A EP1960537A1 EP 1960537 A1 EP1960537 A1 EP 1960537A1 EP 06818281 A EP06818281 A EP 06818281A EP 06818281 A EP06818281 A EP 06818281A EP 1960537 A1 EP1960537 A1 EP 1960537A1
Authority
EP
European Patent Office
Prior art keywords
amplification products
biological sample
different
homologous
amplification
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP06818281A
Other languages
German (de)
English (en)
Inventor
Christoph Gauer
Wolfgang Mann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beckman Coulter Inc
Original Assignee
Advalytix AG
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 Advalytix AG filed Critical Advalytix AG
Publication of EP1960537A1 publication Critical patent/EP1960537A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to a method for determining the genotype of one or more individuals from a biological sample containing nucleic acids of different individuals, in particular a method for determining the copy number of a predetermined sequence, and a kit for determining the genotype of one or more individuals from a biological Sample containing nucleic acids from different individuals.
  • One technique often used in forensics, forensics, or paternity and relationship research to characterize the genotype of one or more individuals is to create a genetic fingerprint.
  • two methods are currently used to generate a genetic fingerprint, namely, the RFLP technique (restriction fragment length polymorphism technique) and the VNTR typification (variable numbers oftandem repeats). Typing).
  • RFLP technique restriction fragment length polymorphism technique
  • VNTR typification variable numbers oftandem repeats.
  • typing DNA is first isolated from biological trace material which contains, for example, blood, saliva, hair with roots, sperm or vaginal secretions. After isolation of the DNA from the biological sample, the RFLP method hydrolyzes the DNA into a variety of lengths of DNA using restriction enzymes, before the individual DNA fragments are separated on an agarose gel lengthwise.
  • Any fluorescence signal present indicates the presence of the sequence corresponding to the probe provided with the corresponding fluorescent label, with the intensity of the fluorescence being able to make a conditional inference to the number of sequence copies in the biological sample.
  • a disadvantage of the aforementioned method is that unwanted cross-hybridization leading to incorrect results can never be completely ruled out.
  • this method is relatively expensive, on the one hand because it is imperative to use fluorescent dyes and, on the other hand, because it requires expensive equipment, such as fluorescence microscopes.
  • Another known method for quantifying nucleic acid sequences is the real-time PCR method, in which, for example, a PCR is carried out with fluorescence-labeled primers and the increase in the fluorescence signal is observed as a function of the number of cycles.
  • the threshold PCR cycle (also known as the threshold cycle) is assigned to the reaction time at which the fluorescence signal is significantly different from the background fluorescence and the PCR product formation proceeds exponentially. This correlates with the initial copy number of the DNA sequence to be amplified. In this way, DNA samples can be relatively quantified by comparison with a series of DNA dilutions.
  • step a) of the method according to the invention can be carried out in any manner known to the person skilled in the art for this purpose.
  • at least two subsets may be taken from the biological sample, which are subsequently diluted with a mutually different dilution factor. It is equally possible to take two aliquots of the sample and concentrate one sample, whereas the other aliquot is either left undiluted or diluted. Any other method which provides at least two subsets of the biological sample, each with a different concentration of the biological sample, can be used in step a).
  • relative quantitative determination of the number of a predetermined sequence in a biological sample in the sense of the present invention means determining whether a biological sample contains fewer, equal to or more copies of a predetermined sequence than a reference sample and absolute quantitative determination of the number of a predetermined sequence in FIG a sample determining which concrete number of copies of the predetermined sequence are contained in the biological sample.
  • the principle of the method according to the invention is therefore based on diluting a subset of the biological sample containing nucleic acids of different individuals until at least a part of the theoretically possible amplification products is no longer obtained, wherein the "failing" amplification products usually those of the in the biological sample in the lowest concentration will be present.
  • a heterogeneous DNA mixture such as a sample containing nucleic acids of different individuals, wherein the nucleic acids of the individual individuals in the biological sample are present in different amounts, the sequences of the nucleic acids of the individual individuals are present in different copy numbers.
  • the experiment allows us to conclude that the DNA present in higher concentrations in the biological sample is attributable to the victim and that the DNA which in the amplification reaction gives two amplification products with a length of 125 and 135 bp is not the victim but attributable to the offender or a third party not involved in the act. Thereafter, the amplification products obtained for the perpetrator or the uninvolved third party can then be further analyzed in a targeted manner in order to conclude, for example by comparison with the data stored in a database, the identity of the offender.
  • the inventive method is simple and inexpensive to perform without costly equipment for the qualitative detection of fluorescence.
  • the method according to the invention is suitable for determining the genotype of one or more individuals from a biological sample containing nucleic acids of different individuals, regardless of the specific number of different individuals.
  • the biological sample contains nucleic acids from at least two but less than 10 different individuals. More preferably, the biological sample contains nucleic acids of at least two, but less than five, most preferably of two or three, and most preferably of exactly two different individuals.
  • the method according to the invention is particularly suitable for forensic examinations, for example in connection with crime investigation.
  • the method of the invention is not limited to this, but can be used for any type of biological sample containing nucleic acids from at least two different individuals.
  • an amplification reaction which is adapted to amplify, for example, 15 different amplification products from the mother's genome, is first carried out with a subset of the undiluted sample and then a dilution series is established with a further subset of the biological sample, the dilution factor between the individual dilution stages, for example, 1: 2. Subsequently, with each dilution step, an amplification reaction is carried out exactly under the same conditions as with the undiluted sample, and the number of different amplification products obtained is determined for each amplification reaction.
  • the at least one amplification reaction is adapted to amplify one or at least two mutually homologous and / or non-homologous sequences which occur only once in the genome of the donor per each allele.
  • conclusions can be drawn on the individual alleles of an individual, so that, for example, the number of individual alleles of an individual in a biological sample comprising nucleic acids of different individuals can be determined.
  • the at least one amplification reaction is adapted to amplify between 1 and 100, preferably between 2 and 20 and more preferably between 5 and 15 mutually homologous and / or non-homologous sequences.
  • the experimental effort is not too big.
  • step b) or step d) of the method according to the invention a PCR adapted to amplify at least two mutually homologous and / or non-homologous sequences is performed, in which one of the number of at least two mutually homologous and / or or non-homologous sequences corresponding number of primer pairs, which are adapted to amplify the at least two mutually homologous and / or non-homologous sequences, is used.
  • An advantage of this embodiment is that in each case only one PCR is necessary for the amplification reaction carried out with the amplification reaction carried out with the undiluted subset of the biological sample and the amplification reaction carried out with the dilution step (s) of the subset of the biological sample, so that the method can be carried out quickly and without large pipetting - Can be carried out effort.
  • An example of a suitable procedure is a multiplex PCR, although any other amplification reaction in which the one or the at least two mutually homologous and / or non-homologous sequences to be amplified can be amplified simultaneously in one reaction can also be used.
  • step b) or step d) a PCR adapted for the amplification of at least homologous and / or non-homologous sequences is carried out, wherein in step a) one of the number of at least two mutually homologous and / or or non-homologous sequences corresponding number of subsets of the biological sample is provided, each subset containing the same amount of biological material, and in step b) or step d) with each of the subsets of a PCR, in each of which a primer pair is used is carried out, wherein the primer pairs used in the various PCRs are adapted to amplify the at least two mutually homologous and / or non-homologous sequences.
  • the presence or absence of amplification products must first be determined.
  • a second, preferably physically and / or chemically measurable parameter must be determined in order to be able to distinguish the individual amplification products from one another to determine the number of different amplification products obtained.
  • the type of the second parameter which distinguishes the individual PCR products, depends essentially on the nature of the one or at least two mutually homologous and / or non-homologous sequences to be amplified.
  • the second parameter is used or as a second distinguishing feature of the individual PCR products, preferably the length of the individual PCR products, so that the determination of the number of different amplification products obtained on the examination for presence or absence of PCR products and the determination of the length of the comprises individual PCR products, wherein the number of different amplification products obtained corresponds to the number of amplification products of differing length obtained.
  • a suitable method for this is, for example, capillary electrophoresis.
  • the second distinguishing feature or the second parameter is preferably the determination of differing sequence, which is usually restricted to one nucleotide in SNP segments.
  • step b) or step d) it has proven to be advantageous to set the parameters in the at least one amplification reaction according to step b) or step d) such that the relative frequency for a positive amplification reaction for the one or each of the at least two mutually homologous and / or non-homologous sequences are each at least substantially equal.
  • the sequence to be amplified of the nucleic acids of the different individuals contained in the biological sample if they are present in an equal amount, amplified with the same effectiveness, so that from the elimination of an amplification product from a certain dilution It can be reliably concluded that the elimination of the amplification product is due to the fact that the DNA of the corresponding individual in the biological sample is present in a correspondingly smaller amount than that of the other individuals, and the omission is not merely based on the fact that the effectiveness of the Amplification reaction for this amplification product was lower than that for another amplification product even with the same amount of DNA.
  • the binding affinity of the individual PCR primers to their primer binding sites and the other parameters of the PCR, in particular the number of cycles and the temperature control, such that the relative frequency for a positive amplification reaction of the at least one amplification reaction for each of the to be amplified mutually homologous and / or non-homologous sequences between 0.2 and less than 1, preferably between 0.4 and 0.6, and particularly preferably about 0.5.
  • the above values for the relative frequency to be set for a positive amplification reaction of the at least one amplification reaction for each of the sequences are not fixed sizes, but in particular depends on the number of used in the PCR start copies. The greater the number of starting copies, the lower the effectiveness of the at least one amplification reaction should be set in order to eliminate the amplification products for the nucleic acid present in low DNA concentration in the biological sample from a relatively low dilution stage. This dependence of the effectiveness to be set on the number of starting copies, ie the number of cells used or the number of copies used, is shown in FIG.
  • the dilution factor to be selected for the subset of the biological sample depends, in particular, on the concentration of the nucleic acids in the biological sample and can easily be determined by one skilled in the art within the scope of experiments which are usual in the art. However, it has proven to be advantageous in principle to use the subset of the biological sample in a ratio of between 1: 1 and 1: 1000, preferably between 1: 1 and 1: 100, more preferably between 1: 1 and 1:10 and most preferably between 1: 1 and 1: 2.
  • the characterization of the amplification products it is possible to use all techniques known to the person skilled in the art which permit a conclusion as to the genotype of the corresponding individual.
  • the characterization of the amplification products may include determining the relative number of one or more alleles of a predetermined sequence.
  • any method known to the person skilled in the art for this purpose can be used. For example, this can be done by carrying out at least one amplification reaction under the same conditions as in step b) of the method according to the invention with a reference sample and the number of different amplification products obtained with this at least one amplification reaction with the reference sample with the number of only part of the Aliquots of amplification products obtained is compared.
  • step b) it is also possible to carry out at least one amplification reaction under the same conditions as in step b) with a reference sample to characterize the amplification products and the number of different amplification products obtained with this at least one amplification reaction with the number of different obtained for all subsets Compare amplification products.
  • the inventive concept it is proposed to compare the number of different amplification products obtained for only a portion of the subsets and / or the number of different amplification products intended for all subsets of the biological sample with at least one frequency distribution for the characterization of the amplification products, the frequency distribution eg. by carrying out the same and the same reaction conditions separately as in step b).
  • at least one amplification reaction wherein in the at least one amplification reaction the same amount of starting material as in step a) was used with at least two different reference samples, the at least two different reference samples each having a known, mutually different copy number of the predetermined sequence, and then determining the number of different amplification products obtained per reference sample was / is obtained.
  • a particularly reliable determination of the relative or even absolute number of alleles of a predetermined sequence in each of the individuals of which nucleic acids are contained in the biological sample is possible.
  • multiple determination of the PCR can also be carried out with one or more dilution stages of the biological sample in order to determine, for example, the relative or absolute number of the predetermined sequence from the comparison of the average of the numbers of different amplification products obtained in the individual determinations to close in the biological sample. It is equally possible to carry out a multiple determination of the PCR with one or more dilution stages of the biological sample and to compare the mean value of the number of a specific, for example allele-specific amplification product obtained with the mean of the number of another specific, for example allele-specific amplification product obtained.
  • At least one primer pair which are adapted, in at least one PCR, one or at least two mutually homologous bi) a reference sample with a known genotype and preferably with a copy number known with respect to a predetermined sequence and / or b2) the result at least one amplification reaction with a reference sample performed under the same conditions as prescribed in the protocol according to d), the reaction conditions being such that the at least one amplification product was formed with a probability of between 20% and less than 100% , and / or b) at least one frequency distribution, which by separately carrying out the same and under the same reaction conditions as in the protocol d) prescribed at least one amplification reaction with at least two different reference samples, wherein the at least two different reference sample n each have a known, mutually different copy number of a predetermined sequence, and subsequent determination of the number of different amplification products obtained per reference sample was obtained, and c) optionally PCR buffer and d) a protocol for performing the at least one PCR in a) and, if appropriate
  • At least two different subsets of different concentrations of at least two under the same conditions as the subsets of the biological sample to be examined are subjected to at least one amplification reaction, even the absolute copy number of the predetermined sequence in the nucleic acid of an individual contained in the biological sample may be subjected to at least one reference sample getting closed.
  • a subset of a concentrated mixed sample containing fetal cells and maternal blood in a ratio of 1: 1000 was subjected to PCR with a primer pair, the primer pair being adapted to amplify one STR sequence of chromosome 21, respectively.
  • an amplification product for a healthy individual homozygous for chromosome 21, an amplification product, for a healthy, heterozygous individual for chromosome 21, two amplification products, for an individual with monoallelic trisomy, an amplification product, for an individual with biallelic trisomy, two amplification products and for one individual triallelic trisomy expected three amplification products.
  • nucleic acid from a third individual is still present in the biological sample, since then two individuals only have one allele would be present in the sample. This is very unlikely.
  • the nucleic acids of the two individuals can now be further characterized as described above.

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Abstract

La présente invention concerne un procédé de détermination du génotype d'un ou de plusieurs individus à partir d'un échantillon biologique qui contient des acides nucléiques provenant de différents individus, et en particulier un procédé de détermination du nombre de copies d'une séquence prédéterminée, dans lequel on réalise d'abord au moins une réaction d'amplification sur au moins deux portions de concentration différente de l'échantillon biologique, on détermine ensuite le nombre des produits d'amplification différents contenus dans chacune des deux portions ou plus pour les comparer les unes aux autres et on caractérise enfin les produits d'amplification qui n'étaient contenus que dans une portion définie et/ou les produits d'amplification qui étaient contenus dans toutes les portions. La présente invention concerne en outre un kit qui permet la mise en oeuvre du procédé selon l'invention.
EP06818281A 2005-12-12 2006-10-24 Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus Withdrawn EP1960537A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005059227A DE102005059227A1 (de) 2005-12-12 2005-12-12 Verfahren zur Bestimmung des Genotyps aus einer biologischen Probe enthaltend Nukleinsäuren unterschiedlicher Individuen
PCT/EP2006/010245 WO2007068305A1 (fr) 2005-12-12 2006-10-24 Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus

Publications (1)

Publication Number Publication Date
EP1960537A1 true EP1960537A1 (fr) 2008-08-27

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Application Number Title Priority Date Filing Date
EP06818281A Withdrawn EP1960537A1 (fr) 2005-12-12 2006-10-24 Procede de determination du genotype a partir d'un echantillon biologique qui contient des acides nucleiques provenant de differents individus

Country Status (5)

Country Link
US (1) US20110124517A1 (fr)
EP (1) EP1960537A1 (fr)
JP (1) JP2009518051A (fr)
DE (1) DE102005059227A1 (fr)
WO (1) WO2007068305A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE449193T1 (de) * 2006-04-12 2009-12-15 Medical Res Council Verfahren zur bestimmung der kopienummer
CA2710807C (fr) * 2008-03-11 2015-09-08 Kyeong Man Hong Procede d'evaluation de nombre de copies de chromosome, gene ou sequence nucleotidique specifique reposant sur l'utilisation d'un jeu snp
DE102008019132A1 (de) * 2008-04-16 2009-10-22 Olympus Life Science Research Europa Gmbh Verfahren zur quantitativen Bestimmung der Kopienzahl einer vorbestimmten Sequenz in einer Probe
JP2012513217A (ja) * 2008-12-22 2012-06-14 セルラ・インコーポレイテッド 対立遺伝子、ゲノムおよびトランスクリプトームを検出する方法および遺伝子型決定パネル
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

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006750A1 (fr) * 1993-09-03 1995-03-09 Cellpro, Incorporated Procede de quantification du nombre de cellules contenant une sequence d'acide nucleique selectionnee dans une population heterogene de cellules
US6180349B1 (en) * 1999-05-18 2001-01-30 The Regents Of The University Of California Quantitative PCR method to enumerate DNA copy number
DE10059776A1 (de) * 2000-12-01 2002-07-18 Adnagen Ag Trisomie 21-Diagnostik-Kit
EP1229128A1 (fr) * 2001-01-31 2002-08-07 Boehringer Mannheim Gmbh Nouveau procédé pour la détermination du génotype
DE10242359A1 (de) * 2002-09-12 2004-03-25 Alopex Gmbh Verfahren zur Amplifikation genetischer Informationen
DE102004036285A1 (de) * 2004-07-27 2006-02-16 Advalytix Ag Verfahren zum Bestimmen der Häufigkeit von Sequenzen einer Probe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007068305A1 *

Also Published As

Publication number Publication date
US20110124517A1 (en) 2011-05-26
WO2007068305A1 (fr) 2007-06-21
DE102005059227A1 (de) 2007-06-14
JP2009518051A (ja) 2009-05-07

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