WO2000066772A1 - Procede de detection par dhplc d'une difference de genotype et/ou de sequence d'adn - Google Patents
Procede de detection par dhplc d'une difference de genotype et/ou de sequence d'adn Download PDFInfo
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- WO2000066772A1 WO2000066772A1 PCT/FR2000/001198 FR0001198W WO0066772A1 WO 2000066772 A1 WO2000066772 A1 WO 2000066772A1 FR 0001198 W FR0001198 W FR 0001198W WO 0066772 A1 WO0066772 A1 WO 0066772A1
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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
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
Definitions
- the subject of the present invention is a method of detecting by denaturing high performance liquid chromatography (DHPLC) a difference in genotype or sequence between a sample of natural or artificial DNA heterozygous or homozygous reference for a genomic fragment of interest and a sample of natural or artificial heterozygous or homozygous DNA to be tested for said genomic fragment of interest.
- the invention also includes the use of this method for the genetic diagnosis of a disease as well as for the search and / or identification of polymorphism or of a sequence variation for a genomic fragment of interest.
- heteroduplex and homoduplex, or between heteroduplex makes it possible in particular to detect the presence of mutant DNA from amplification product resulting from a denatured and then rehybridized mixture of amplification product of wild DNA and homozygous mutant DNA. , or resulting from the amplification product of a denatured then rehybridized heterozygous mutant DNA.
- the DHPLC method thus described does not make it possible to detect the presence of a polymorphism, mutation or genetic variation of sequence carried by a fragment of heterozygous DNA and not by a reference heterozygous DNA for said fragment, when the two types of heterozygotes give the same DHPLC profile.
- the mutation located in the Notch 3 gene in the heterozygous state gives the same DHPLC profile as a non-functional polymorphism located in the same DNA fragment also in the heterozygous state, whereas the DHPLC method described in the prior art does not not allowing them to be distinguished. This mutation is more difficult to identify by sequencing.
- the method according to the present invention has the principle of multiplexing the amplification products of heterozygous DNA samples giving the same profiles by mixing said amplification products, then denaturing and rehybridizing them, and then analyzing on DHPLC in denaturing condition, the amplification products thus rehybridized.
- the mixture of two amplification products of two heterozygous DNA samples of the same genotype will give the same heterozygote and therefore the same heteroduplex which will give the same profile in DHPLC as the amplification products of the heterozygous DNA samples tested separately.
- the mixture of two amplification products of two different heterozygous DNA samples will generate in addition new species of heteroduplex which will cause the profile corresponding to the mixture to differ from the profiles of the two amplification products of DNA samples heterozygotes tested separately.
- the present invention relates to a method of detection by high performance liquid chromatography in denaturing condition (DHPLC) of a difference in genotype or sequence between a sample of reference heterozygous DNA for a genomic fragment of interest and a sample of Heterozygous DNA to be tested for said genomic fragment of interest, characterized in that it comprises the following stages: a) denaturation: ⁇ ) when the number of DNA samples to be tested is equal to one, of a mixing an amplification product of a sample of said reference heterozygous DNA with an amplification product of a sample of said heterozygous DNA to be tested; or ⁇ ) when the number of DNA samples to be tested is at least equal to two, of a mixture of each amplification product of a sample of the heterozygous DNAs to be tested with an amplification product of the sample reference heterozygous DNA; b) hybridization of the denatured mixture of the amplification products obtained in step a); c) producing the DHPLC profile for the hybrid mixture of the a
- high performance liquid chromatography method in denaturing condition By “high performance liquid chromatography method in denaturing condition”, called DHPLC, applied here to a sample containing DNA, is meant in the present description a separation method by high performance liquid chromatography (HPLC) in which the separation DNA contained in the sample is carried out under condition of at least partial thermal denaturation of this DNA.
- HPLC high performance liquid chromatography
- Such methods make it possible in particular to differentiate, from the separation profile obtained (absorbance curve as a function of the retention time of the detected compound), hereinafter referred to as DHPLC profile, from homoduplex compounds to heteroduplex compounds (Kuklin et al., Genetic Testing, 1 (3), 201-206, 1997;
- DNA fragment WAVE TM from the company Transgenomic Inc. (Santa Clara, CA, USA) comprising a high resolution polymeric matrix of the DNASep TM type in partially denaturing condition or methods incorporating equivalent systems for automatic analysis of fragments of DNA comprising high resolution HP type columns (Hewlett Packard) such as the systems offered by the company Varian (Hélix System, TM Prostar).
- DNA denaturation of a DNA sample is meant the process aimed at separating two strands of paired DNA, not requiring the breaking of covalent bonds.
- DNA denaturation occurs and is carried out in a thermal amplitude field which can vary depending on the concentration of certain compounds facilitating the destabilization of hydrogen bonds, such as cations, and / or according to the base composition (content of GC bases) and / or also according to the degree of pairing of the strands to be denatured.
- the optimal conditions for separation of the homoduplex DNA from the heteroduplex DNA by the DHPLC method such as in particular the optimal analysis temperature and / or the buffer gradient used for separation.
- the methods making it possible to optimize these parameters as a function of the genomic fragment of interest chosen are well known to those skilled in the art and will not be developed here (cf. for example: Taylor et al., Application note N ° 101 , Transgenomic Inc., Santa Clara, CA, USA; Kuklin et al., Genetic Testing, 1 (3), 201-206, 1997).
- genomic fragment of interest we mean on the one hand:
- any artificial double-stranded nucleic acid fragment, DNA or RNA resulting from unnatural hybridization between a first strand of DNA or RNA of a eukaryotic or prokaryotic cell, a virus or an organelle with a second strand of DNA or RNA of a eukaryotic or prokaryotic cell, of a virus or of an organelle complementary to the first strand on which fragment a mutation, a polymorphism or in general a sequence variation is sought relative to a reference nucleic acid sequence for said artificial nucleic acid fragment; said genomic fragment of interest being used as DNA or double stranded RNA template for amplification, knowledge of said sequence of reference allowing the development of primers from which said natural or artificial fragment of interest, can be amplified.
- the amplification reaction carried out from this template will be preceded by a reverse transcription step (such as for example the technique called RT-PCR).
- genomic fragments of interest corresponding to natural fragments of double-stranded nucleic acid
- genomic fragments of interest corresponding to natural fragments of double-stranded nucleic acid
- genomic fragments of gene coding for a genetic marker, a regulatory part or a promoter part of a gene or for a protein in particular the transcribed part of a gene and its translated part, in particular the genes whose presence of mutation directly or jointly determines with one or more other genes a pathology or the risk of a pathology, or even the genomic fragments whose knowledge of all of their polymorphisms in a given population makes it possible to identify classes of population exhibiting or which may exhibit a specific response to a given therapeutic compound.
- genomic fragments of interest containing a transgenic sequence the detection of which will be the subject of the invention, the DNAs to be tested being able to come from a transgenic organism, such as transgenic animals or plants, or from cells or transformed or recombinant microorganisms.
- genomic fragments of interest corresponding to artificial fragments of double-stranded nucleic acid there may be mentioned, for example, but not limited to, the fragments resulting from the unnatural hybridization between:
- a first strand of DNA or RNA of a human cell which can be transformed, coding for a gene of interest with a second strand of DNA or RNA complementary to the first strand from a non-human animal cell (rat, mouse, etc.) and the sequence of which, coding for the equivalent gene, has a sufficient degree of identity to allow hybridization of the two strands;
- a first strand of DNA or RNA from a human cell transformed by a pathogenic virus HIV, hepatic viruses, HBV, HCV, HDV, or others
- a pathogenic virus HBV, hepatic viruses, HBV, HCV, HDV, or others
- a first strand of DNA or RNA from an animal, plant, yeast, bacteria, mycoplasma, parasite or virus strain coding for a gene of interest with a second strand of DNA or RNA complementary to the first strand from another strain of the same organism and whose sequence has a sufficient degree of identity to allow hybridization of the two strands;
- a first strand of DNA or RNA of an organelle of the mitochondria or chloroplast type coding for a gene of interest derived from a first individual with a second strand of DNA or RNA complementary to the first strand derived from, or prepared from another individual and whose sequence has a sufficient degree of identity to allow hybridization of the two strands;
- a first strand of DNA or RNA from an animal or plant cell encoding a gene of interest with a second strand of DNA or RNA complementary to the first strand from a transformed cell of the same animal or plant, for example from a transgenic animal or plant (rat, mouse, plant of industrial interest, etc.) and whose sequence, coding for the same gene has been modified and has a sufficient degree of identity to allow hybridization of the two strands; or
- heterozygous DNA sample or heterozygous template for said genomic fragment of interest is meant a double stranded DNA sample natural or artificial comprising double-stranded DNA having at least one polymorphism, mutation or sequence variation between the different strands that make it up or make it up.
- samples of natural heterozygous DNA for a genomic fragment of interest very particularly preferred are samples of DNA from diploid organisms, in particular samples of heterozygous DNA from eukaryotic cells or diploid viruses, and of which said samples genomic fragments of interest correspond to natural fragments of double-stranded nucleic acid, which may or may not include fragments of transgenic sequence (or transgene or heterologous sequence).
- sample of heterozygous DNA or natural or artificial reference heterozygous matrix for said genomic fragment of interest is intended to denote a sample of heterozygous DNA originating from a natural or artificial genomic fragment for said genomic fragment of interest capable of forming after amplification, an amplification product which, after denaturation and hybridization, forms a mixture of homoduplex and heteroduplex whose DHPLC profile obtained serves as a reference profile.
- amplification product of a DNA sample is meant the product, or a sample of said product, obtained after one or more cycles (denaturation, hybridization and elongation) during which the quantity of target DNA corresponding to the fragment genomics of interest has been increased, such as for example the products obtained by elective amplification techniques by polymerase chain reaction (PCR) or its variants (NASBA TM, etc.).
- PCR polymerase chain reaction
- NASBA TM polymerase chain reaction
- the expression “production of the DHPLC profile” is understood to mean the step comprising the separation of the DNAs forming homoduplexes or heteroduplexes at least partially denaturing conditions and the establishment of the DHPLC profile.
- difference in genotype or sequence is intended to denote any variation in the sequence of a nucleic acid to be tested with respect to a reference nucleic sequence, said variation resulting from the deletion, insertion or substitution of at least one nucleotide of the reference nucleic sequence, the nucleic acid to be tested being able to come from a genomic fragment of the same population as the reference nucleic acid (intraspecies), or to be coming from a genomic fragment of the same population as the reference nucleic acid but originating, for example, from a cell of a tissue different from that from which the reference nucleic acid (intertissues within the same species), or else originating from a genomic fragment from a population different from that from which the reference nucleic acid is derived (interspecies, for example Man / animal, except Man, such as Man / mouse, Man / rat, Man / monkey , Male / nematode or Man / Drosophila, Man / virus, or mouse / rat, transformed cell
- the method according to the invention also makes it possible to detect by DHPLC a difference in genotype or sequence for a genomic fragment of interest between a sample of reference heterozygous DNA and at least one sample of heterozygous DNA among several DNA samples. heterozygote to be tested whose amplification products are mixed before carrying out the DHPLC profile.
- the DHPLC method is sensitive enough to detect the presence of heteroduplexes obtained after hybridization different from those obtained for the reference sample even after dilution of these heteroduplexes due to the mixing of the amplification products of all the samples d Heterozygous DNA to be tested with the amplification product of the reference heterozygous DNA sample.
- This variant of the process according to the invention can advantageously be used in applications where it is usually necessary to systematically carry out the sequencing of a given DNA fragment in order to determine the presence of one or more polymorphisms, of mutation or sequence variation for a large number of DNA samples, insofar as this variant makes it possible to avoid the sequencing of a group of DNA samples by a single DHPLC analysis when the profile obtained for the mixture of the amplification products from this group has a profile identical to the DHPLC profile of the amplification product of heterozygous DNA of reference for the genomic fragment of interest. Indeed, the only sequencing of the reference heterozygous DNA is then sufficient to identify the genotypes of all the samples of heterozygous DNA to be tested whose amplification products have been mixed.
- Such a variant can advantageously be used in the field of large-scale diagnosis of a disease of genetic origin, of a predisposition of genetic origin to a given trait or a disease, or even to determine one or more polymorphisms in a given population associated with tolerance or not with a given therapeutic compound.
- Such a variant can also be advantageously used to determine all of the polymorphisms present on one or more genomic fragments of interest in a given population usually requiring systematic sequencing of the DNA fragment or fragments for each of the individuals.
- the detection method according to the present invention is characterized in that the number of heterozygous DNA samples to be tested for the genomic fragment of interest is between 1 and 10, preferably between 1 and 5, ends included , even more preferably equal to 1.
- the invention also includes a detection method according to the invention, characterized in that the nucleic sequence of the genomic fragment of interest is known.
- the term “knowledge of a nucleic sequence” is intended to denote knowledge of at least one fragment of said sequence, or of a fragment located near or overlapping said sequence, making it possible to develop one or a set of several primers from which or which amplification of the genomic fragment of interest of the heterozygous DNA samples to be tested and of reference will be initiated, and if necessary the sequencing of said genomic fragment of interest, and / or the sequencing of said genomic fragment of interest of heterozygous DNA samples to test and reference.
- the invention also relates to a detection method according to the invention, characterized in that the comparison between the DHPLC profile obtained for the amplification product of a sample of heterozygous DNA to be tested and the DHPLC profile obtained for the product d amplification of a sample of said reference heterozygous DNA does not make it possible to detect a difference in genotype or sequence for said genomic fragment of interest between the reference heterozygous DNA and the heterozygous DNA to be tested.
- the invention further relates to a detection method according to the invention, characterized in that the mixing of the amplification products of step a) is carried out from the amplification product of said DNAs obtained separately.
- the reference heterozygous DNA sample and the heterozygous DNA sample to be tested will be amplified separately and then the two amplification products obtained will be mixed before stage a) of denaturation.
- the present invention also comprises a method according to the invention characterized in that the mixture of the amplification products of step a) is obtained directly by simultaneous amplification of said DNA samples.
- the reference heterozygous DNA sample and the heterozygous DNA sample to be tested will be previously mixed and then amplified simultaneously, thus making it possible to directly obtain the mixture of the two amplification products before stage a) of denaturation.
- the heterozygous DNA samples to be tested and the reference heterozygous DNA sample will also be mixed beforehand, then amplified simultaneously before step a) of denaturation.
- the invention relates to a detection method according to the invention, characterized in that the heterozygous DNA samples for a genomic fragment of interest are chosen from DNA samples from diploid organisms, especially from DNA samples from two different tissues of the same diploid organism.
- the invention also relates to a detection method according to the invention, characterized in that the genomic fragment of interest is chosen from the genomic fragments of interest corresponding to natural fragments of double-stranded nucleic acid , said natural fragments optionally comprising a fragment of transgenic sequence.
- the subject of the invention is a method for identifying at least one mutation, a polymorphism or a sequence variation carried by the nucleic sequence of a sample of heterozygous DNA to be tested for a genomic fragment of interest, characterized in that it comprises the following steps: a) detection by DHPLC of a difference in genotype or sequence between a sample of a reference heterozygous DNA whose nucleic sequence is known for said genomic fragment of interest and said heterozygous DNA sample to be tested by a detection method according to the invention; b) sequencing the nucleic sequence of the heterozygous DNA sample to be tested; c) the identification of the mutation, of the polymorphism or of the sequence variation carried by the nucleic sequence of the heterozygous DNA sample to be tested by comparison of its sequence with the nucleic sequence of the reference heterozygous DNA.
- the invention also includes a method for confirming a difference in genotype or sequence between a reference heterozygous DNA sample for a genomic fragment of interest and a heterozygous DNA sample to be tested for said genomic fragment of interest , characterized in that it implements a detection method according to the invention or an identification method according to the invention.
- the methods of detection, identification or confirmation according to the invention may be used for the search for a known or unknown mutation in a genomic fragment of interest or for the genetic diagnosis of diseases linked to the presence of a pathogenic mutation, for the genetic diagnosis of predisposition to diseases linked to the presence of a pathogenic mutation or predisposition to a given trait.
- the invention also comprises the use of a detection method, an identification method or a confirmation method according to the present invention, for the detection of at least one polymorphism , of all polymorphisms in a given population or of any variation of sequence carried by a genomic fragment of interest, in particular for pharmacogenetics and for the constitution of a map of genetic markers or for the demonstration of a transgenic sequence carried by a genomic fragment of interest.
- the method according to the invention constitutes a very significant improvement brought to the detection capacity of DHPLC columns to distinguish different polymorphisms, mutations or sequence variations in the same DNA fragment when these cannot be differentiated without mixing of heterozygous individuals.
- the method according to the invention can also be used systematically for the detection and identification of new polymorphisms, mutations or sequence variations, to ensure more efficient discovery of these polymorphisms, mutations or genetic sequence variations by DHPLC.
- the method according to the invention makes significant savings in cost of sequencing for diagnosis, discovery of polymorphisms, mutations or variations in sequence.
- the sequencing of all heterozygous individuals is no longer necessary to identify all of the polymorphisms, mutations or variations in sequence carried by a DNA fragment and in a given population. Only the sequencing of a heterozygous individual corresponding to each family of different profiles obtained in DHPLC after multiplexing is now necessary to identify all the polymorphisms and / or mutations carried by the different heterozygotes of a population for a given DNA fragment. .
- the method according to the invention allows considerable savings to be made in any known or unknown mutation, sequence or sequence variation detection program in a given population.
- the invention also relates to the use of the methods according to the invention for the detection and identification of inter-tissue variation of nucleic sequence for a genomic fragment of interest.
- the subject of the invention is a method for detecting by high performance liquid chromatography in denaturing condition (DHPLC) a difference in genotype or sequence between a sample of reference heterozygous or homozygous DNA for a genomic fragment.
- DPLC denaturing condition
- artificial homoduplex or heteroduplex is intended to denote a double-stranded DNA, the first strand of which is obtained from a sample of heterozygous or homozygous DNA from a genomic fragment of a diploid or haploid cell, such as eukaryotic cells ( animal or plant), parasite, prokaryotic or virus, or organelle such as mitochondria or chloroplast, is hybrid with the second strand of said double stranded DNA, said second strand being from a sample of heterozygous or homozygous DNA a genomic fragment from another species of diploid or haploid cell or from an organelle of the same species but from another individual, or from a cell of the same organism, but from a different tissue .
- artificial homoduplexes or heteroduplexes mention may be made, for example, but not limited to, double-stranded DNA resulting from unnatural hybridization between:
- a first strand of DNA from a human cell which can be transformed, coding for a gene of interest with a second strand of DNA complementary to the first strand from an animal cell (rat, mouse, etc.) and whose sequence, coding for the equivalent gene has a sufficient degree of identity to allow hybridization of the two strands;
- a first strand of DNA from a human cell transformed by a pathogenic virus HIV, hepatic viruses, HBN, HCN, HDN, or others
- a pathogenic virus HBV, hepatic viruses, HBN, HCN, HDN, or others
- a first strand of DNA from an animal or plant cell coding for a gene of interest with a second strand of DNA complementary to the first strand from a transformed cell of the same animal or plant for example from of a transgenic animal or plant (rat, mouse, plant of industrial interest, etc.) and whose sequence, coding for the same gene has been modified and has a sufficient degree of identity to allow hybridization of the two strands ;
- a first strand of DNA or RNA from an animal, plant, yeast, bacteria, mycoplasma, parasite or virus strain coding for a gene of interest with a second strand of DNA or RNA complementary to the first strand from another strain of the same organism and whose sequence has a sufficient degree of identity to allow hybridization of the two strands;
- a first strand of DNA or RNA of an organelle of the mitochondria or chloroplast type coding for a gene of interest derived from a first individual with a second strand of DNA or RNA complementary to the first strand derived from, or prepared from another individual and whose sequence has a sufficient degree of identity to allow hybridization of the two strands; or, in general
- homozygous DNA sample or homozygous template for said genomic fragment of interest is meant a double stranded DNA sample from a diploid organism whose two alleles carrying the genomic fragment of interest are identical, or from 'an organism or micro-organism or virus or haploid organelle comprising a unique sequence for the genomic fragment of interest.
- homozygous DNA sample or homozygous reference matrix for said genomic fragment of interest is meant a homozygous DNA sample for said genomic fragment of interest capable of forming, after amplification, amplification products which after denaturation and hybridization form a mixture of homoduplex whose DHPLC profile obtained serves as a reference profile.
- samples of homozygous DNA for a genomic fragment of interest very particularly preferred are samples of DNA from diploid organism, in particular samples of homozygous DNA from eukaryotic cells or diploid viruses, or also from haploid organism or microorganism, in particular derived from prokaryotes, viruses or organelle such as mitochondria or chloroplasts, and of which said genomic fragments of interest correspond to natural fragments of double-stranded or single-stranded nucleic acid , which may or may not include fragments of transgenic sequence (or transgene or heterologous sequence).
- RNA sample corresponding to the transcription of the genomic fragment of interest (in particular an mRNA) or of a genomic fragment of interest from an organism whose genome consists of RNA (mono- or double-stranded)
- RT-PCR reverse transcription
- a detection method characterized in that the number of heterozygous or homozygous DNA samples to be tested for the genomic fragment of interest is between 1 and 10, preferably between 1 and 5, ends included , preferably equal to 1; - a detection method according to the invention, characterized in that the nucleic sequence of the genomic fragment of interest is known;
- a detection method characterized in that the comparison between the DHPLC profile obtained for the amplification product of a heterozygous or homozygous DNA sample to be tested and the DHPLC profile obtained for the amplification product of a sample of said heterozygous or homozygous reference DNA does not make it possible to detect a difference in genotype for said genomic fragment of interest between the reference heterozygous or homozygous DNA and the heterozygous or homozygous DNA to be tested;
- step a) A detection method according to the invention, characterized in that the mixing of the amplification products of step a) is carried out from the amplification product of said DNAs obtained separately;
- step a) A detection method according to the invention, characterized in that the mixture of the amplification products of step a) is obtained directly by simultaneous amplification of said DNA samples;
- heterozygous or homozygous DNA samples for a genomic fragment of interest are chosen from DNA samples from a diploid or haploid organism or microorganism; or
- genomic fragment of interest is chosen from the genomic fragments of interest corresponding to natural fragments of double-stranded or single-stranded nucleic acid which may or may not contain fragments of transgenic sequence.
- the invention also relates to the use of the methods according to the invention for the characterization of the genetic variability of haploid organisms, such as mycoplasmas, bacteria, viruses, yeasts, parasites or organelles.
- haploid organisms such as mycoplasmas, bacteria, viruses, yeasts, parasites or organelles.
- the mixing of the amplification products from step a) of the process according to the invention can be carried out either from the amplification product of the DNA samples obtained separately, or from the amplification products obtained directly by amplification simultaneous DNA samples.
- a sample of DNA (or RNA which will then be transcribed into DNA) of an individual or of a particular strain will be taken as reference for the production of artificial heteroduplexes.
- the difference in genotype or sequence thus detected can then be identified by sequencing the DNA fragment of interest and comparison of the sequence obtained with the reference sequence.
- the characterization of the genetic variability of these haploid organisms or microorganisms can be applied to the preparation of an entire map of genetic markers of their entire genome or of a particular gene.
- the methods according to the invention may make it possible to identify one or more markers whose presence is linked directly or indirectly, for example, but not limited to, a sensitivity or resistance character of a virus, a parasite, a yeast or a bacteria in a given medium (nutritive or other), an antibiotic agent, a natural phenomenon like cold, heat, need for water, sun or darkness , or its infectivity, its virulence, its rate of recombination, its adaptability to its hosts, its duration of latency, survival, the evolution of the disease or any other applications of which the making of these cards could be the subject .
- the invention also relates to the use of the methods according to the invention for the characterization of the interspecies genetic variability for diploid or haploid organisms or microorganisms.
- the methods of detection, or of identification when followed by a sequencing step, according to the invention can indeed make it possible to study the sequence variations for a given gene which has been conserved during evolution .
- the mixing of the amplification products from step a) of the process according to the invention can be carried out either from the amplification product of the DNA samples obtained separately, or from the amplification products obtained directly by amplification simultaneous DNA samples for a genomic fragment of interest corresponding to a gene conserved for example in mice, rats, monkeys, dogs, humans, nemotodes or drosophila.
- a sample of DNA (or RNA which will then be transcribed into DNA) of a particular species will be taken as a reference for the production of artificial heteroduplexes.
- the characterization of the genotype or sequence differences for the gene of interest can be used in order to identify a link between sequence and evolution of a function encoded by this gene.
- the invention also relates to the use of the methods according to the invention for the characterization of the genetic variability between different pure strains of the same diploid species.
- the methods of detection, or of identification when followed by a sequencing step, according to the invention can indeed make it possible to study the variations in sequences for one or more particular genes known as a function of a given character.
- the mixing of the amplification products of step a) of the method according to the invention can be carried out either from the amplification product of the DNA samples obtained separately, or from the amplification products obtained directly by amplification simultaneous DNA samples for a genomic fragment of interest corresponding to a gene or several genes which are assumed to be linked to a given trait, or to a given pathology.
- the detection or identification methods according to the present invention may make it possible to detect or identify the variations in the sequences of one or more genes which are assumed to be linked to the loss or hair growth, hair color, size, etc. (to a given benign phenotype) or to a pathology such as atherosclerosis for example.
- the pure murine strain C3H is resistant when the pure murine strain C57B16 is sensitive to atherosclerosis.
- the mixture of the DNA amplification products of these strains, DNA corresponding to a gene suspected of being linked to atherosclerosis will make it possible to characterize the variation of the gene as a function of the affection.
- the invention also relates to the use of the methods according to the invention for the characterization of the genetic variability among different genomes 1 organelles such as chloroplasts or mitochondria in plants in animals.
- Organelles are indeed extranuclear entities with chromosomal DNA distinct from nuclear nucleic acid. Their DNA codes for particular genes which, when mutated, can cause disease.
- the transmission of organelles like mitochondria and therefore of the characters they carry is non-Mendelian. For example in humans, we inherit the mitochondria from its mother, the sperm not contributing to the constitution of the mitochondria of the egg and therefore as a consequence of the mitochondria of the developed being. Certain diseases caused by mutations in mitochondrial DNA are therefore called maternal transmission diseases.
- the organelle genome is haploid
- the characterization of the variability of its sequence in a given species like man can only be done by making artificial heteroduplexes.
- step a) of the process according to the invention can be carried out in the same way either from the amplification product of the DNA samples from mitochondria obtained separately, or from the products of amplification obtained directly by simultaneous amplification of DNA samples from mitochondria for a genomic fragment of interest corresponding to a gene or several genes which are assumed to be linked to a given pathology or characteristic.
- the detection or identification methods according to the present invention may make it possible to detect or identify the variations in the sequences of one or more genes of these organelles which are assumed to be linked to a given trait such as photosynthesis for chloroplasts or cellular respiration for mitochondria, or to one or more genes whose expression is linked to mitochondrial diseases with maternal transmission, or to map genetic markers of these organelles.
- the invention also relates to the use of the methods according to the invention for the characterization of inter-tissue genetic variability.
- the mixing of the amplification products of step a) of the method according to the invention can be carried out in the same way either from the amplification product of the DNA samples of healthy tissue and of diseased tissue (which they either homozygous or heterozygous) obtained separately, or from the amplification products obtained directly by simultaneous amplification of the samples of said DNA for a genomic fragment of interest corresponding to a gene or several genes which are assumed to be linked to the pathology.
- FIG. 1A Theoretical explanatory diagram showing that two amplification products obtained from two samples of heterozygous DNA (a: reference DNA and b: DNA to be tested) presenting between them a genotypic difference and giving separately the same DHPLC profile (FIG. 1A) gives, if they are mixed beforehand (multiplexing), a DHPLC profile different from the profile obtained for the reference DNA sample (FIG. 1B) thus highlighting the presence of a genotypic difference between these two heterozygous DNA samples.
- FIG. 2 A corresponds to the profile of a heterozygote for a pathogenic CADASIL mutation (position B in the genomic fragment of interest).
- Figures 2B and 2C correspond to the profiles obtained for two heterozygotes for another benign polymorphism (position A) also present in the same genomic fragment of interest. This polymorphism is itself non-pathogenic (position A).
- Figure 2D corresponds to the profile of a wild homozygote for the same amplified genomic fragment.
- Figures 3A to 3C represent the profiles obtained by DHPLC produced on the WAVE TM system.
- Figures 3 A and 3B correspond to the profiles obtained separately with the two heterozygotes for position A.
- FIG. 3C corresponds to the profile obtained with the mixture of the two heterozygotes of FIGS. 3A and 3B.
- Figures 4A to 4C represent the profiles obtained by DHPLC produced on the WAVE TM system.
- FIG. 4A corresponds to the profile obtained with a heterozygote for position A.
- FIG. 4B corresponds to the profile obtained with the heterozygote for the mutation
- Figure 4C corresponds to the profile obtained with the mixture of the two heterozygotes in Figures 4 A and 4B.
- Figures 5A and 5B represent the comparison between two profiles obtained with two types of heterozygous mixtures by DHPLC produced on the WAVE TM system.
- FIG. 5A represents the profile obtained with a mixture of a heterozygote for position A and a heterozygote for the CADASIL mutation in position B in the genomic fragment of interest.
- FIG. 5B represents the profile obtained with a mixture of two heterozygotes for the non-pathogenic polymorphism present in position A in the same genomic fragment of interest.
- the DNA samples from wild heterozygous and homozygous individuals for exon 3 of the human Notch 3 gene used come from the group of Elisabeth Tournier-Lasserve (INSERM U25, Necker Hospital, Paris, France):
- 10 ng of genomic DNA are amplified by PCR with two primers specific for exon 3 of human Notch 3 according to a conventional protocol and which generates, after 35 amplification cycles, a product of 224 base pairs.
- the size and quality (no parasitic band) of the amplified fragments can be checked on a 2% agarose gel by electrophoresis before the test on DHPLC.
- the genomic fragment of interest in this example relates to a fragment of
- This fragment is amplified by PCR for each of the DNA samples from individuals to be diagnosed for the presence of polymorphism in position A or B or both, then denaturation of these PCR products at 95 ° C for 3 min and slow renaturation (or hybridization) by slow cooling of ⁇ 1.6 ° C./minute for 45 minutes for each of the samples before testing them on a DHPLC column under semi-denaturing conditions (Transgenomic machine and SARASEP TM column) according to the method described after.
- 66 ° C temperature determined empirically and which is close to Tm (temperature for which 50% of the amplified fragment is denatured) of the amplified fragment of 224 base pairs.
- Tm temperature for which 50% of the amplified fragment is denatured
- empirically is meant to mean that a fusion curve of the amplified fragment of interest has been established on WAVE TM by determining the retention times of a wild-type amplification product for this fragment as a function of different temperatures between 60 ° C and 70 ° C and according to a gradient defined thanks to the retention time of said amplification product subjected to the universal gradient recommended by the manufacturer Transgenomic (routine procedure).
- Buffer A 0.1 molar of triethylammonium acetate (TEAA).
- Buffer B 0.1 molar TEAA / 25% acetronitrile (HPLC grade).
- One of the two heterozygotes for position A is used as the reference heterozygous DNA for the two mixtures tested.
- the two heterozygotes of position A on the one hand and a heterozygote of position A with the heterozygote of position B are mixed on the other hand.
- FIGS. 2A to 2D show that the three heterozygotes (two for position A, one for position B) taken separately have the same profile and that they cannot be distinguished on the basis of their respective profile obtained in DHPLC.
- FIGS. 3A to 3C show that the two profiles obtained with the two heterozygotes for position A and that obtained with their mixture are identical confirming that the two heterozygotes mixed are identical.
- Figures 4A to 4C show that the profile obtained with the mixture of the heterozygote for position A and the heterozygote for the CADASIL mutation (position B) ( Figure 4A) is different from the profiles obtained with the two heterozygotes taken separately (figures 4 A and 4B) which are identical to each other.
- the mixture of heterozygotes therefore made it possible to distinguish in DHPLC and at a lower cost than the sequencing of an individual carrying the CADASIL mutation of a heterozygous individual for a non-pathogenic polymorphism.
- Figures 5A and 5B show the supe suposition of the profiles presented in figures
- DHPLC makes it possible to distinguish and / or confirm a difference in genotype between two heterozygous DNA samples whose profiles obtained separately are identical by mixing beforehand the amplification products of these two DNA samples according to the process of the invention, and thus the DHPLC used according to the process of the invention is a powerful tool for the genetic diagnosis of this type of mutation or for the demonstration or the discovery of polymo ⁇ hism.
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU44126/00A AU4412600A (en) | 1999-05-04 | 2000-05-04 | Method for detecting by dhplc a difference of dna genotype and/or sequence |
EP00925382A EP1173617A1 (fr) | 1999-05-04 | 2000-05-04 | Procede de detection par dhplc d'une difference de genotype et/ou de sequence d'adn |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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FR99/05651 | 1999-05-04 | ||
FR9905651A FR2793262B1 (fr) | 1999-05-04 | 1999-05-04 | Procede de detection par dhplc d'une difference de genotype et/ou de sequence entre des adn naturels ou artificiels heterozygotes ou homozygotes pour un fragment genomique d'interet et son application |
Publications (1)
Publication Number | Publication Date |
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WO2000066772A1 true WO2000066772A1 (fr) | 2000-11-09 |
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PCT/FR2000/001198 WO2000066772A1 (fr) | 1999-05-04 | 2000-05-04 | Procede de detection par dhplc d'une difference de genotype et/ou de sequence d'adn |
Country Status (4)
Country | Link |
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EP (1) | EP1173617A1 (fr) |
AU (1) | AU4412600A (fr) |
FR (1) | FR2793262B1 (fr) |
WO (1) | WO2000066772A1 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2817559A1 (fr) * | 2000-12-06 | 2002-06-07 | Genodyssee | Procede de determination d'un ou plusieurs polymorphisme(s) fontionnel(s) dans la sequence nucleique d'un gene "candidat" fonctionnel preselectionne et ses applications |
US6455692B1 (en) | 1998-08-04 | 2002-09-24 | Transgenomic, Inc. | Method of concentrating polynucleotides using MIPC |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5795976A (en) * | 1995-08-08 | 1998-08-18 | The Board Of Trustees Of The Leland Stanford Junior University | Detection of nucleic acid heteroduplex molecules by denaturing high-performance liquid chromatography and methods for comparative sequencing |
WO1999054498A1 (fr) * | 1998-04-17 | 1999-10-28 | Astrazeneca Ab | Methode de detection d'un desequilibre allelique |
-
1999
- 1999-05-04 FR FR9905651A patent/FR2793262B1/fr not_active Expired - Fee Related
-
2000
- 2000-05-04 WO PCT/FR2000/001198 patent/WO2000066772A1/fr active Search and Examination
- 2000-05-04 AU AU44126/00A patent/AU4412600A/en not_active Abandoned
- 2000-05-04 EP EP00925382A patent/EP1173617A1/fr not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5795976A (en) * | 1995-08-08 | 1998-08-18 | The Board Of Trustees Of The Leland Stanford Junior University | Detection of nucleic acid heteroduplex molecules by denaturing high-performance liquid chromatography and methods for comparative sequencing |
WO1999054498A1 (fr) * | 1998-04-17 | 1999-10-28 | Astrazeneca Ab | Methode de detection d'un desequilibre allelique |
Non-Patent Citations (5)
Title |
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LIU W ET AL: "DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC) USED IN THE DETECTION OF GERMLINE AND SOMATIC MUTATIONS", NUCLEIC ACIDS RESEARCH,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 26, no. 6, 1 January 1998 (1998-01-01), pages 1396 - 1400, XP002911657, ISSN: 0305-1048 * |
MANSUKHANI ET AL: "Convenient, nonradioactive heteroduplex based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes", DIAGNOSTIC MOLECULAR PATHOLOGY,US,NEW YORK, NY, vol. 6, no. 4, August 1997 (1997-08-01), pages 229 - 237-237, XP002109367 * |
NOLLAU ET AL: "Methods for detection of point mutations", CLINICAL CHEMISTRY,US,AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY. WINSTON, vol. 43, no. 7, 1997, pages 1114 - 1128-1128, XP002109368, ISSN: 0009-9147 * |
OEFNER P J ET AL: "COMPARATIVE DNA SEQUENCING BY DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC)", AMERICAN JOURNAL OF HUMAN GENETICS,US,NEW YORK, NY, October 1995 (1995-10-01), pages COMPLETE01, XP002916094, ISSN: 0002-9297 * |
THOMPSON C T ET AL: "CYTOGENETIC PROFILING USING FLUORESCENCE IN SITU HYBRIDIZATION (FISH) AND COMPARATIVE GENOMIC HYBRIDIZATION (CGH)", JOURNAL OF CELLULAR BIOCHEMISTRY. SUPPLEMENT,US,A.R. LISS, NEW YORK, NY, no. SUPPL. 17G, 1 January 1993 (1993-01-01), pages 139 - 143, XP000612761, ISSN: 0733-1959 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6455692B1 (en) | 1998-08-04 | 2002-09-24 | Transgenomic, Inc. | Method of concentrating polynucleotides using MIPC |
FR2817559A1 (fr) * | 2000-12-06 | 2002-06-07 | Genodyssee | Procede de determination d'un ou plusieurs polymorphisme(s) fontionnel(s) dans la sequence nucleique d'un gene "candidat" fonctionnel preselectionne et ses applications |
WO2002046459A2 (fr) * | 2000-12-06 | 2002-06-13 | Genodyssee | Procede de determination d'au moins un polymorphisme fonctionnel dans la sequence des nucleotides d'un gene candidat preselectionne et applications dudit procede |
WO2002046459A3 (fr) * | 2000-12-06 | 2003-03-13 | Genodyssee | Procede de determination d'au moins un polymorphisme fonctionnel dans la sequence des nucleotides d'un gene candidat preselectionne et applications dudit procede |
Also Published As
Publication number | Publication date |
---|---|
EP1173617A1 (fr) | 2002-01-23 |
FR2793262B1 (fr) | 2003-01-31 |
AU4412600A (en) | 2000-11-17 |
FR2793262A1 (fr) | 2000-11-10 |
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