WO2014009578A1 - Méthode pour l'obtention du profil génétique d'un individu par analyse de loci de chromosome x - Google Patents

Méthode pour l'obtention du profil génétique d'un individu par analyse de loci de chromosome x Download PDF

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WO2014009578A1
WO2014009578A1 PCT/ES2013/000170 ES2013000170W WO2014009578A1 WO 2014009578 A1 WO2014009578 A1 WO 2014009578A1 ES 2013000170 W ES2013000170 W ES 2013000170W WO 2014009578 A1 WO2014009578 A1 WO 2014009578A1
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seq
locus
dxs
loci
dna
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PCT/ES2013/000170
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Spanish (es)
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Maria CASTAÑEDA FERNÁNDEZ
Adrián ODROZIOLA MARTÍNEZ
Maria Teresa ZARRABEITIA CIMIANO
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Universidad De Cantabria
Fundación Marqués De Valdecilla
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Publication of WO2014009578A1 publication Critical patent/WO2014009578A1/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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the invention relates to a method for obtaining the genetic profile of an individual comprising the analysis of 4 or more STRs loci, up to a total of 8 loci, all located on the X chromosome, in a DNA extract of said individual by a multiplex amplification reaction.
  • the present invention is also directed to a kit comprising the pairs of primers for the implementation of the method of the invention. Both the method and the kit described here are applicable in the identification of individuals in the area of forensic genetics, as well as in population genetics, anthropology and biomedicine.
  • the X chromosome comprises 155 Mb and represents 5% of the total genetic material of the cells. It contains approximately 1 100 genes. Only 1.7% of this chromosome encodes functional proteins, presenting a high content of repetitive non-coding DNA (Ross et al., 2005 Nature 434: 325-337). Part of this fraction are short regions repeated in tandem or "short tandem repeats" (STR). To date, more than fifty STRs have been described on the X chromosome whose analysis is fundamentally of interest among others, in Forensic Genetics and in the aforementioned disciplines (Gusmao et al., 2012 Methods Mol. Biol. 830: 57-71 ). The analysis of microsatellite markers called STRs (Short Tandem Repeats) is nowadays the reference technique for obtaining genetic profiles aimed at identifying individuals, determining kinship or crime.
  • STRs Short Tandem Repeats
  • X-chromosome STR analysis is a complementary tool of high interest (Zarrabeitia et al., 2002 Forensic Sci. Int. 129 (2): 85-89; Szibor et al., 2003 Int. J. Legal Med. 1 17: 67-74; Liu et al, 2008 Int. J. Legal Med. 122: 261-265). Likewise, it has demonstrated great utility in the identification of individuals.
  • the haplotype of the paternal X and Y chromosome can be detected respectively in the female and male offspring.
  • This X chromosome that the mother transmits may be an exact copy of any of its chromosomes or it may have undergone recombination between the two X chromosomes and be different from the initial copy that the woman possesses (Wakefield et al., 2006 Encyclopedia of life sciences ).
  • the haplotype of the father's X chromosome will be identical to one of the paternal grandmother's chromosomes, so if at least one allele of each marker analyzed by the grandmother does not match that of the granddaughter, biological paternity can be ruled out. These analyzes have a high probability of exclusion. In turn, in those cases in which the grandmother is also unavailable, her genotype can be reconstructed through the study of the siblings of the alleged father (Szibor et al, 2007 Forensic Sci.
  • the microsatellites of the X chromosome complement the autosomal STR analysis, since if the daughter becomes pregnant, in her female offspring there will be no allele other than those present in said parent. Its usefulness would stand out in terms of excluding a supposed incest. While due to the limitations of inclusion of the X chromosome STRs, it is necessary to consider that the X chromosome haplotype can be shared by other members of the same family (Szibor et al., 2007 Forensic Sci. Int: Genet. 1: 93-97; Szibor et al., 2003 Int. J. Legal Med. 1 17: 67-74).
  • X-chromosome microsatellite studies are especially useful in identifying a female profile in evidence that contains a mixture of male and female DNA.
  • the alleles of the female sample can be completely masked by the alleles of the male sample, only if the woman is homozygous and coincides in all loci with the man, very unlikely event. Therefore, these analyzes are useful in solving criminal cases in which we give as an example, it is necessary to demonstrate the presence of female epithelial or vaginal cells, in the body of the alleged aggressor or in objects with which there are been in touch
  • the degradation of DNA is characterized by a general fragmentation of the genetic material, where DNA fragments can be around 240 bp, and these reactions require larger fragments (table 2), for the analysis of all STRs loci that include commercial kits.
  • Highly degraded DNA is a characteristic of most of the samples that are analyzed in criminal genetic laboratories, in kinship diagnoses and also of those obtained from biological samples contained in paraffin in health centers. In these cases the absence of results or - -
  • the first of these reactions has been specifically designed to analyze the DXS7423, GATA31E08, DXS6789 and DXS 101 loci, from fragments that exceed 169 bp base pairs. While the second reaction allows the analysis of loci DXS7133, DXS7424, GATA165B12 and DXS8378, even from fragments that exceed 1 1 1 bp.
  • Table 4 also shows a comparative study of the base pair sizes of the amplicons corresponding to miniSTR loci of the X chromosome in the main multiplex reactions of the prior art.
  • the miniSTRs are the products resulting from the redesign of primers, which join regions as close as possible to the structure of the STR, with the purpose of obtaining amplicons of reduced size.
  • Table 4 List of the main miniSTR loci of the prior art. The base pair size of each amplicon is detailed when analyzed by the main multiplex reactions.
  • a main aspect of the invention relates to a method for obtaining the genetic profile of an individual comprising the analysis, in a DNA extract from said individual, by means of a multiplex amplification reaction of at least 4 STRs loci of the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 where
  • At least one of the 4 STR loci is selected from the group formed by the loci
  • At least two of the 4 STRs loci are selected from the group consisting of the DXS7132, DXS6801, DXS 10075 and DXS 10079 loci.
  • the invention provides a kit comprising pairs of specific primers to amplify at least 4 STR loci selected from the group formed by loci DXS6799, DXS10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS10079, wherein a) at least 1 of the 4 STRs loci is selected from the group consisting of the DXS6799, DXS 10074, DXS6789 and DXS6809 loci; Y
  • the invention relates to a set of primer pairs comprising at least 4 primer pairs aimed at amplifying 4 STR loci of the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 wherein a) at least one of the 4 STRs loci is selected from the group consisting of the DXS6799, DXS 10074, DXS6789 and DXS6809 loci; Y
  • At least two of the 4 STRs loci are selected from the group consisting of the DXS7132, DXS6801, DXS 10075 and DXS 10079 loci.
  • the use of the set of primer pairs of the invention is contemplated to obtain the genetic profile of an individual, to determine kinship relationships between individuals or to identify human remains,
  • the invention relates to a pair of primers selected from the group consisting of the following oligonucleotide pairs - SEQ ID NO: 1 and SEQ ID NO: 2 for locus DXS6799,
  • Figure 1 corresponds to a graph called electropherogram showing the genetic profile of an individual for the STRs analyzed by the set of primers of the invention using an automatic AB310 Genetic Analyzer (Applied Biosystems ® ) sequence analyzer.
  • panels can be observed, each corresponding to those pairs of primers marked with the same fluorescent locus.
  • Each panel shows the intensity of the signal detected in the Relative Fluorescence Units (RFU, relative fluorescence unit) on the Y axis;
  • the X axis corresponds to the size in base pairs. So, - -
  • the first panel corresponds to the results obtained for the products of STRs amplified with primers marked with 6-FAM TM (directed to the loci DXS6799 and DXS 10074, the second panel to those marked with VIC TM (DXS7132 and DXS6801), the third to NED TM (loci DXS6789 and DXS6809) and fourth panel results for loci STRs labeled with the fluorochrome PET ® (DXS 10075 and DXS 10079) are shown.
  • Figure 2 is a graphic representation showing the design of the set of primer pairs called miniX.
  • the boxes represent the amplified sizes for each locus Loci marked with different color are presented in different gray scales
  • the respective tides are indicated in the right part of the figure
  • Figure 3 is a comparison of the The method obtained by miniX and Decaplex, in the analysis of the loci shared by both reactions, from 24 paraffin samples.
  • Figure 4 is a comparison of the yield obtained by miniX and Sextaplex, in the analysis of the loci shared by both reactions, from 24 paraffin samples.
  • Said feature allows the application of this invention in the resolution of complex paternity cases from highly degraded DNA samples more effectively than commercial kits developed to date for the analysis of the aforementioned X chromosome loci.
  • the terms “genetic profile” and “genetic fingerprint” have the same meaning and can therefore be used interchangeably throughout the description.
  • the “genetic profile” is a set of sequences of characteristic bases of the genetic material that allow differentiating an individual from another. STR loci
  • STRs sequences or "STRs loci” means those DNA sequences, also called microsatellites, that contain 4 bp repeats (Butler JM, Biotechniques. 2007).
  • the analysis of the STRs loci in the genome presents multiple applications that include, but are not limited to, obtaining genetic profiles, kinship diagnoses, determining the origin of a sample or tissue, identification of cadavers, population genetics studies, tests of zygosity in twins, monitoring of bone marrow transplants, evaluation of the traceability of biological samples of human origin, identification of mixtures present in a sample and determination of the number of contributors of human origin to a mixture and their contribution relative to the mixture.
  • PCR corresponds to the polymerase chain reaction acronym whereby millions of copies of the desired DNA regions can be obtained. It is characterized by the use of pairs of primers that delimit the region from which millions of copies will be made, which is also known as "amplifying DNA" during PCR.
  • the PCR is composed of a certain number of cycles, in turn composed of three phases in which the DNA strands are separated, the primers are joined and the new DNA strands are elongated. In each cycle, if the reaction efficiency is 100%, exponential growth of the DNA fragments subject to amplification occurs.
  • multiplex amplification reaction is understood as the PCR reaction in which more than one DNA sequence is amplified in the same reaction, by using two or more pairs of primers in a single tube together with the rest. of the reaction reagents in order to simultaneously amplify multiple DNA sequences.
  • oligonucleotide refers to the sequence of nucleotide bases linked by phospho-diester bonds, usually not greater than 50 nucleotides.
  • primer or “first” or “oligonucleotide” (“oligo”) is understood as the nucleotide sequence from which DNA polymerase initiates the synthesis of a new DNA molecule.
  • the primers are short nucleotide sequences, approximately 15-24 nucleotides in length that can be aligned with a strand of target DNA thanks to the complementarity of bases to form a hybrid between the primer and the target strand of DNA. Then, the DNA polymerase enzyme can extend the primer along the DNA strand. Methods for preparing and using primers are described, for example in Sambrook et al., 2001 and Ausubel et al., 1999.
  • primer pair or “first pair” is understood as the set of two primers which, when used in the same amplification or PCR reaction, allow multiple copies of a DNA target sequence to be obtained.
  • Each of the primers hybridizes with the target sequence, so that the bounded nucleotide sequence is amplified by each pair of primers.
  • the extent of the primers during the PCR cycles determines the 2 N exponential multiplication of the nucleotide sequence bounded by the primers, N being the number of cycles of the PCR reaction.
  • biological sample means any matter that contains a nucleic acid, for example, DNA.
  • DNA or “genomic DNA” is understood as the genetic material of living organisms that controls inheritance and is located in the nucleus of cells.
  • DNA extract is understood when, after subjecting the biological sample to a method of extraction, separation, purification or cloning of DNA, among others, it is obtained as a result either dry, in solution, bound or not to other molecules, adhered or not to various substances or beds, matter in which the DNA is in a greater relative proportion with respect to the rest of the molecules present, compared to the biological starting sample.
  • DNA extract refers to DNA extracted from any biological sample that contains human DNA, whether from a living or dead individual, fetus, organs, tissues or cells.
  • the term "individual” refers to a human being of any age or race, who in the context of the present invention may be alive or dead.
  • the inventors have designed a pair of primers, henceforth, "pairs of primers of the invention” or “miniX”, which allows obtaining the genetic profile of the STRs loci (DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801 , DXS 10075 and DXS 10079) of an individual, even from highly degraded DNA samples, and is applicable even to complex kinship analysis.
  • MiniX is characterized by allowing a high number of STR loci to be analyzed by small amplified amplifiers. So, it has been specifically designed to enable the amplification of 8 STRs loci, from DNA fragments that exceed 220 bp. It stands out for being the only system currently available that allows analyzing all the STRX DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 loci, in a single multiplex reaction. In addition, it includes amplifiers smaller than those available in the state of the art in 6 of the 8 STR loci included.
  • the primer pairs have been designed so that their application allows obtaining the genetic profile of an individual that comprises the combination of at least 4 to 8 STR loci.
  • the designed primer pairs have the additional technical characteristic that, if desired, they can be used simultaneously in combinations of 4 or up to 8 primer pairs in a multiplex amplification reaction to obtain a genetic profile composed of 4 or more STRs loci up to a total of 8 STR loci whose analysis of high interest in Forensic Genetics and Population Genetics, without limiting this interest to these areas.
  • STRs loci analyzed in the present invention as well as the pairs of primers designed for identification are shown in Table 5. This same table details the minor to major allele of the STR that will be considered by this analysis (column: alleles) as well. as the maximum and minimum possible size for each STR.
  • Example 1 details the procedure followed in the design of the set of primer pairs of the invention. - -
  • Example 1 the design of the primer pairs of the present invention was carried out by searching the flanking sequences at each STR locus. Subsequently, the Perlprimer program was used. Once the primer pairs were obtained, a polymerase amplification reaction was carried out on a DNA extract using said primer pairs after which, the genetic profile of the individual to whom the DNA extract belonged reliably was obtained, precision and with a discriminating power superior to the genetic profiles obtained by other methods from highly degraded DNA samples, as illustrated in example 2.
  • Table 5 Summary of the sequence of primers in the 'to 3 direction 'designed for the amplification of each STR in the combination called as miniX (column: sequence 5 ' - 3 ' ). The variation (rs945048 A / T) of the degenerate primers sequence is underlined and the G base added to the sequence appears in bold for the DXS 10075 locus.
  • a main aspect of the invention relates to a set of primer pairs, hereafter "set of primer pairs of the invention", comprising at least 4 primer pairs aimed at amplifying 4 loci STRs of the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079, where - -
  • At least one of the 4 STRs loci is selected from the group consisting of the DXS6799, DXS10074, DXS6789 and DXS6809 loci; Y
  • the set of primer pairs of the invention comprises at least 5, 6, 7 or 8 specific primer pairs for the STR loci of the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079.
  • primer pairs are selected from the group consisting of the following oligonucleotide pairs:
  • the invention relates to a pair of primers selected from the group consisting of the following oligonucleotide pairs:
  • uses of the set of primer pairs of the invention include, but are not limited to, obtaining genetic profiles, kinship diagnoses, determining the origin of a sample or tissue, identification of cadavers, population genetics studies, evidence of Zygosity in twins, monitoring of bone marrow transplants, evaluation of the traceability of biological samples of human origin, identification of mixtures present in a sample, determination of the number of contributors of human origin to a mixture and their contribution relative to the mixture.
  • the use of the set of primer pairs of the invention is contemplated to obtain the genetic profile of an individual, to determine kinship relationships between individuals or to identify human remains.
  • the present invention is based on the design of a pair of primers that allow obtaining the genetic profile of an individual based on a combination of at least 4 STRs.
  • Another main aspect of the invention relates to a method, hereinafter "method of the invention", to obtain the genetic profile of an individual comprising the analysis, in a DNA extract from said individual, by means of a multiplex amplification reaction, of at least 4 STRs loci from the group consisting of the DXS6799, DXS10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 loci, where a) at least one of the 4 STRs loci is selected from group formed by loci DXS6799, DXS 10074, DXS6789 and DXS6809; Y
  • At least two of the 4 STRs loci are selected from the group consisting of the DXS7132, DXS6801, DXS 10075 and DXS 10079 loci.
  • the primer pairs have been designed in such a way that it is possible to use them in the analysis of 4 or more, up to a total of 8 STRs loci, (DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079).
  • STRs loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079.
  • the set of primer pairs aimed at obtaining the genetic profile of an individual is directed to the analysis of at least 5, 6, 7 or 8 STR loci of the group formed by the loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079.
  • the primer pair employed in the multiplex amplification reaction specific to each of the STR loci analyzed is the oligonucleotide pair shown in the sequences.
  • the implementation of the method of the invention requires obtaining a DNA extract that will ultimately come from a biological sample that will be adequately treated to obtain said DNA extract.
  • the DNA extract will come from the individual whose genetic profile is to be obtained, or from the individuals whose kinship relationship wants to be determined.
  • the present invention is especially indicated to resolve those kinship relationships in which the biological samples of some of the people involved are not available.
  • the method of the invention is directed to the identification of human remains, the DNA extract will come from the human remains that are to be identified.
  • the term "human remains” includes any biological sample from a human being, said human residue being able to be preserved in formol, frozen, dried, fixed in paraffin, in a state of decomposition, degradation and / or rot, between others.
  • samples from which DNA can be obtained are, without limitation, biological fluids (blood, saliva, urine, sperm, etc); epidermis, dandruff, hair, feces, a vaginal sample, a tissue sample, burned tissues, etc.
  • biological fluids blood, saliva, urine, sperm, etc
  • epidermis dandruff, hair, feces, a vaginal sample, a tissue sample, burned tissues, etc.
  • the most suitable samples for obtaining DNA extracts useful in the identification of human remains include, among others, root hairs, compact bone, tooth, soft tissues and blood.
  • the DNA extract comes from a sample of blood, hair, saliva, epidermis, sperm, dandruff, ashes, a vaginal sample and a tissue sample,
  • the fraction of DNA suitable for the implementation of the invention is nuclear DNA. DNA extraction may be performed using any method known to those skilled in the art (Sambrook et ai, 2001 "Molecular Cloning: A Laboratory Manual", 3rd ed, Cold Spring Harbor Laboratory Press, NY, Vol . 1.
  • kits - -3) including without limitation, density gradient centrifugations, two-phase extraction using aqueous phenol or chloroform with ethanol, column chromatography, methods based on the ability of DNA to bind on glass surfaces and / or silicates, as preparations of diatomaceous earth or glass beds, using kits - -
  • the DNA extract is quantified and normalized to obtain equal amounts of DNA in each sample in case the amount of DNA is sufficient for it.
  • the DNA extract from the biological sample After obtaining the DNA extract from the biological sample, it is subjected, at least, to a multiplex amplification reaction using the set of primer pairs of the invention.
  • the amplification of the different STRs loci requires specific reaction conditions and procedures for each of the pairs of primers used, which can be achieved by systematic variation of each parameter.
  • the number of cycles and the alignment temperature used in the PCR reaction must be suitable for obtaining results by each of the first set of primers. Parameters such as the concentration of primers are specific to each primer and have been adjusted in the validation of the primer set.
  • the primers offer results in a wide range of concentrations, although the optimal concentrations for each pair of primers are shown in Table 6.
  • a multiplex amplification reaction requires a series of reagents, including, but not limited to, the template DNA, the enzyme DNA polymerase, at least two pairs of primers (each primer being each pair complementary to one of the two strands of the DNA), deoxynucleotide triphosphates (dNTPs), magnesium chloride (MgC12), reaction buffer and optional additives, which can be added separately by mixing in the laboratory or purchased previously mixed, as is the case of Multiplex PCR master Mix (Qiagen, Hilden, Germany), or Kapa2GTMFast Multiplex PCR kit (apa Biosystems, Inc., USA), to which the primer pairs are added in the appropriate concentrations and the template DNA.
  • dNTPs deoxynucleotide triphosphates
  • MgC12 magnesium chloride
  • the PCR is carried out in a thermal cycler that performs the cycles in the exact times and temperatures programmed, such as the hybridization temperature or the melting temperature.
  • the melting temperature of the multiplex amplification reaction comprising oligonucleotides SEQ ID NO: 1 to SEQ ID NO. 17 is from 57 to 63 ° C as calculated using the Perlprimer software (http://perlprimer.sourceforge.net/).
  • the priming of the primers participating in said multiplex amplification reaction can be carried out in order to be able to subsequently detect the amplified fragments.
  • the marking of amplification products can be carried out by conventional methods. Said marking can be direct, for which fluorophores can be used, for example, Cy3, Cy5, fluorescein, alexa, etc., enzymes, for example, alkaline phosphatase, peroxidase, etc., radioactive isotopes, for example, 33P, 1251, etc., or any other marker known to the person skilled in the art.
  • said marking can be indirect through the use of chemical, enzymatic methods, etc .
  • the amplification product may incorporate a member of a specific binding pair, for example, avidin or streptavidin conjugated with a fluorochrome (locus), and the probe binds to the other member of the specific binding pair, for example, biotin (indicator), the reading being carried out by fluorimetry, etc.
  • the amplification product may incorporate a member of a specific binding pair, for example, an anti-digoxigenin antibody conjugated to an enzyme (locus), and the The probe binds to the other member of the specific binding pair, for example, digoxigenin (indicator), etc., the enzyme substrate is transformed into a luminescent or fluorescent product and the reading is carried out by chemo-luminescence, fluorimetry, etc.
  • the marking of the amplification product is carried out by marking, at one of its ends, one of the oligonucleotides of each primer pair.
  • the oligonucleotide mapping is selected from the group consisting of a radioisotope, a fluorescent material, digoxigenin and biotin.
  • 5-FAM 5-carboxyfluorescein
  • 6-FAM t-FAM tetrachlorinated analog
  • HEX 6-FAM hexachlorinated analog
  • TAMRA 6- carboxytetra
  • the amplification products or amplicons can be separated.
  • Virtually any conventional method can be used within the framework of the invention to separate products from -
  • Techniques for separating amplification products are widely described in the state of the art, such as in Sambrook et al., 2001 (cited ad supra). Techniques for separating amplification products are, for example, submerged electrophoresis with Methafor gels, polyacrylamide gels electrophoresis, capillary electrophoresis, etc.
  • the size of the separated fragments is identified, for which any of the methods of identification of amplification fragments known in the state of the art can be used, such as hybridization with labeled probes (for example with a fiuorophore) that will be detected by a detector and processed by a computer system, staining, for example, with ethidium bromide, silver staining, etc.
  • labeled probes for example with a fiuorophore
  • staining for example, with ethidium bromide, silver staining, etc.
  • the invention relates to a kit useful for the implementation of the method of the invention, comprising the set of primer pairs of the invention comprising 4 or more primer pairs aimed at amplifying the STRs loci (DXS6799 , DXS 10074, DXS6789, DXS6809, DXS7132, DXS680I, DXS 10075 and DXS 10079).
  • kit hereinafter kit of the invention, comprising pairs of specific primers to amplify at least 4 STR loci selected from the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS680I, DXS 10075 and DXS 10079, where at least 1 of the 4 STRs loci is selected from the group consisting of the DXS6799, DXS 10074, DXS6789 and DXS6809 loci; and at least two of the 4 STRs loci are selected from the group consisting of the DXS7132, DXS6801, DXS 10075 and DXS 10079 loci.
  • kit hereinafter kit of the invention, comprising pairs of specific primers to amplify at least 4 STR loci selected from the group formed by loci DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS680I,
  • the kit of the invention comprises at least 5, 6, 7 or 8, pairs of primers specific for the STR loci of the group formed by loci DXS6799, DXS10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS10075 and DXS10079 .
  • the primer pair used in the multiplex amplification reaction specific to each of the STR loci analyzed is the oligonucleotide pair shown in the sequences.
  • the kit of the invention in addition to comprising the set of primer pairs of the invention, may optionally include the reagents necessary to carry out the multiplex amplification reaction, including, without limiting to, deoxynucleotides triphosphate (dNTPs), divalent and / or monovalent ions, a buffer solution (buffer) that maintains the proper pH for the functioning of DNA polymerase, DNA polymerase or mixture of different polymerases, etc.
  • dNTPs deoxynucleotides triphosphate
  • buffer solution buffer
  • the kit of the invention does not comprise the reagents necessary to practice the method of the invention, these are commercially available and can be found as part of a kit.
  • any commercially available kit containing the reagents necessary to carry out an amplification reaction can be used successfully in the practice of the method of the invention. As shown in the examples, in the case of the present invention these reagents are previously mixed in the Multiplex PCR master Mix reagent (Qiagen, Hilden, Germany). - -
  • the priming of the primers can be carried out in order to be able to subsequently detect the amplified fragments.
  • the kit of the invention may comprise the first set of primer pairs of the invention wherein one of the oligonucleotides of each pair of primers is labeled at one of its ends or, alternatively , may comprise the reagents necessary to carry out the priming of the primers.
  • the kit of the invention further comprises the reagents necessary to label the nucleotide pairs.
  • one of the oligonucleotides of each pair of primers is labeled at one of its ends.
  • the markers used in the oligonucleotide mapping select from the group consisting of a radioisotope, a fluorescent material, digoxigenin and biotin.
  • the fluorescent material is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-FAM, t-FAM tetrachlorinated analog (TET), 6-FAM hexachlorinated analog (HEX), 6- carboxytetramethylrodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein (JOE), NED (ABI), Cy-3, Cy-5, Cy-5.5, fluorescein-6- isothiocinate (FITC) and tetramethylrodamine-5-isothiocinate (TRITC).
  • the kit of the invention is useful in the implementation of the kit of the invention is useful in the implementation of the kit of the invention.
  • Example 1 the validation of miniX is performed, for use in human identification.
  • Example 1 the optimization of the primer sets and the parameters of the PCR reactions is detailed, as well as the studies of the ratio between the height of heterozygotes (PHR), stutter bands (stutter bands) ) and sensitivity.
  • PHR heterozygotes
  • stutter bands stutter bands
  • Example 2 In the second example, the validation of the method of the invention comprising the multiplex amplification reaction is shown for use in the analysis of highly degraded DNA samples, in order to demonstrate that the invention is suitable for use. In this type of samples.
  • miniX is a suitable tool for obtaining human genetic profiles.
  • Saliva samples were taken from 439 healthy individuals: 365 residents in Santander (Cantabria, Spain) grouped into 1,116 families and 74 inhabitants of Vega de Pas (Cantabria, Spain) grouped into 22 families. In all cases the families were constituted at least by paternal grandfather, mother and son. DNA extraction and quantification
  • DNA extracts from population samples were obtained by extraction by Prep Mini Spin kit (GE Healthcare Spain, Barcelona,
  • Quantification of DNA extracts was performed using Quant-iT TM dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA).
  • the Perlprimer program http://perlprimer.sourceforge.net/) was used to design new primers that amplify 8 STR loci (DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079), based on the reference sequences whose access numbers to GeneBank are shown in Table 7.
  • the flanking regions to the STRs analyzed were studied using SNPblast www.ncbi.nlm.nih.gov/SNP/snpblastByChr.html) in order to avoid variable positions in the junction region of all designed primers.
  • the amplifiers range from 101 to 219 bp, to cover a large number of the alleles contained in the reference publications detailed in Table 1, made based on the information collected in ChrX-STR.org 2.0 [http: // www .chrx-str.org].
  • each amplifier product was analyzed mixed with 9 ⁇ of Hi-Di TM Formamide (Applied Biosystems ® , Foster City, CA) and 0.3 ⁇ of GeneScan TM 500 LIZ ® Size Standard (Applied Biosystems ® , Foster City, AC). After the denaturation of the amplified (5 minutes at 95 ° C) they were cooled (5 minutes at 4 ° C) and separated in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems ® , Foster City, CA), Electrophoresis results were analyzed using Genmapper software version 3.2 (Applied Biosystems ® , Foster City, CA).
  • Table 7 shows a wide range of alleles for each locus included in miniX.
  • the alleles described in the main population groups have been considered according to the information collected in ChrX-STR.org 2.0 [http://www.chrx-str.org].
  • 37 primers were designed from which those capable of producing amplifiers of the smallest possible size were chosen, which in no case exceeded 220 bp, whose melting temperatures were between 57.5 and 60.5 ° C and that hybridize in regions lacking SNPs ⁇ Single Nucleotide Polymorphism) and INDELS (Insertions / deletions) described to date in NCBI (http://www.ncbi.nlm.nih.gov/snp/).
  • the degenerate primers (degenerate primers) design for the DXS 10075 locus was necessary to allow hybridization to the primer binding site, because an SNP (rs945048 A / T) was found close to the repeat unit of said STR .
  • SNP rs945048 A / T
  • first phase of optimization singleplex PCR amplifications of each locus were performed.
  • second optimization phase various sets of multiplex primers were tested.
  • the allele ranges of adjacent loci in size were spaced in order to avoid overlaps between alleles corresponding to different loci to ensure proper genotyping.
  • duplex A DXS6799 and DXS 10074; duplex B: DXS6789 and DXS6809; duplex C: DXS7132 and DXS6801 and duplex D: DXS 100075 and DXS 10079) were selected.
  • primers were designed so that a total of 8 STRs loci could be analyzed in a single multiplex reaction: (DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079).
  • the optimization of the amplification parameters such as the optimal concentration of primers, hybridization temperature, number of cycles and extension time, was carried out by studying the electro ferograms in which the height of the peaks was evaluated, the balance in heterozygous thereof and incomplete adenylation.
  • concentration of each pair of primers was tested between 0.04 ⁇ and 0.6 ⁇ , so that in each case, below the optimum concentration an increase in the height of the peaks was observed as that the concentration of primers was increased. While at the same time, in concentrations higher than the one considered optimal, an increase in both imbalance in heterozygosis and incomplete adenylation was detected.
  • the hybridization temperature of the multiplex PCR reaction was then optimized.
  • the increase in hybridization temperature produced a gradual decrease in incomplete adenylation, but also in the height of the peaks.
  • the best results were obtained at the hybridization temperature of 58 ° C.
  • miniX has proven to be a balanced and specific multiplex reaction, capable of amplifying 8 STRs loci, from 0.5 ng of template DNA (Figure 1), by the following amplification conditions: an initial denaturation cycle during 15 minutes at 95 ° C, 30 cycles of 30 s at 94 ° C, 90 s at 58 ° C and 60 s at 72 ° C, followed by the final 60 minute extension cycle at 60 ° C.
  • MiniX has proven to be a balanced and specific multiplex reaction, capable of amplifying 8 STRs loci. In this way miniX is the only reaction capable of analyzing simultaneously - -
  • miniX ftability has been verified by concordance studies between the genetic profiles determined by this system and those obtained through widely used and validated pre-existing systems such as Decaplex and Sextaplex (Gusmao et al., 2009 Int. J. Legal Med. 123 ( 3): 227-234; Casta ⁇ eda et al., 2012 J. Forensic Sci. 57 (1): 192-195). In this sense, 100% concordance was observed between the results of the three reactions compared. Sextaplex shares 6 of the STR loci included in miniX (DXS 10074, DXS6789, DXS6809, DXS6801, DXS 10075 and DXS 10079).
  • miniX is a system of high robustness and reliability in obtaining genetic profiles, which makes them a useful tool for use in human identification.
  • the small size of its amplifiers smaller in its totality at 219 bp suggests that the present invention has a high analysis capacity of the DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 loci.
  • Validation of the method of the invention comprising a multiplex amplification reaction using sets of miniX primer pairs for use in the analysis of highly degraded DNA samples - -
  • K562 control DNA (Promega® Corporation, USA) was used to perform sensitivity assays. This sample was quantified using Quant-iT TM dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications and was diluted to 0.5 ng / ⁇ .
  • Saliva samples were taken from 439 healthy individuals: 365 residents in Santander (Cantabria, Spain) grouped into 1,116 families and 74 inhabitants of Vega de Pas (Cantabria, Spain) grouped into 22 families. In all cases the families were constituted at least by paternal grandfather, mother and son. -
  • DNA extracts from population samples were obtained by extraction by Prep Mini Spin kit (GE Healthcare Spain, Barcelona, Spain) and those from paraffin by cobas® DNA Sample Preparation Kit (Roche Roche Diagnostics SL, Barcelona, Spain). Quantification of DNA extracts was performed using Quant-iT TM dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA).
  • the amplification was performed in a GeneAmp ® 9700 PCR System thermocycler (Applied Biosystems, Foster City, CA), under the following conditions: an initial denaturation cycle for 15 minutes at 95 ° C, 30 cycles of 30 s at 94 ° C, 90 at 58 ° C and 60 s at 72 ° C, followed by the final 60 minute extension cycle at 60 ° C.
  • stuttering bands which are allelic products that differ from the true allele only in a unit of repetition.
  • the determination of stuttering alleles in both homozygous and heterozygous individuals that differed in size by more than 4 bp was made by calculating the percentage of the stutter allele's height relative to the actual peak's height.
  • the calculation of the relative intensity of heterozygous peaks (“Peak Height Ratio" or PHR) was done by dividing the height of the smallest of the peaks by the height of the largest of the heterozygotes of each locus.
  • control DNA 562 was analyzed in duplicate (Promega ® Corporation, USA). DNA concentrations of 1 ng, 500 pg, 200 pg, 100 pg, 75pg, 62.5 pg, 50 pg, 25 pg, 20 pg, 10 pg and 5pg were used.
  • the least amount of DNA that allowed to obtain complete profiles by duplicate miniX analysis was 20 pg. Even amounts of DNA in the range of 10 to 20 pg, allowed the analysis of 5 STR loci (a fixed core of 4 STRs loci: DXS6799, DXS6789, DXS6809 and DXS 10079; and DXS 10075 or DXS 10074, depending on the replica ) by alleles whose intensity was in the range 42-466 RFUs. While the remaining 2 STR loci (DXS7132, DXS6801) presented allelic losses due to stochastic effects or did not offer results at more than 40 RFUs in any of the duplicates. Comparative study of the design strategies of the primer sets of the present invention with respect to prior art methods
  • the size of the amplicons of a multiplex STR system is critical for obtaining - -
  • table 1 1 the discrimination power obtained by miniX, Decaplex and Sextaplex is detailed, after the analysis of the loci shared between miniX and said reactions, from highly degraded DNA fragments: miniX vs. Decaplex (DXS6789, DXS6809 and DXS7132); and miniX vs. sextaplex (DXS 10074, DXS6789, DXS6809, DXS6801, DXS 10075 and DXS10079).
  • the values obtained correspond to the analysis of DNA fragments of 220 bp.
  • the intensity of the signal of an allele is related to its ability, so that genotyping based on alleles whose height does not exceed 50-100 RFUs (Relative Units of Fluorescence) is usually not performed. That is why the performance in the analysis of highly fragmented DNA samples has been evaluated based on two variables: A) the percentage of samples in which each STR has been genotyped, discarding those alleles of height less than 50 RFUs; and B) the percentage of samples in which the genotyping corresponds to alleles of height greater than 200 RFUs, indicating extra reliability of the result.
  • miniX has a higher overall performance than Decaplex in 2 of the 3 shared loci, with the exception of the DXS7132 locus, which has been genotyped by Decaplex in 92% of the samples compared to 88 % in the case of miniX. Although, in this locus, said 4% difference corresponds to alleles whose height does not exceed 200 RFUs. On the other hand, miniX shows a global performance superior to Sextaplex in the 6 shared STR loci and a higher percentage of alleles above 200 RFUs (Figure 4).
  • the percentage values of stuttering alleles of mean, median, standard deviation, minimum and maximum values of the method of the invention are adjusted to those obtained by other research groups in the validation of other STRs loci analysis systems of Chromosome X (Asamura et al., 2006 Int. J. Legal Med. 120: 174-181, in the validation of other multiplex reactions of autosomal STRs such as I-DNA-1 (Odriozola et al., 201 1 Int. J. Legal Med. 125 (5): 685-94), I-DNA-2 (Odriozola et al., 2012 Int. J. Legal Med. 126 (1): 167-72) and geneRESMPX-3 (Schlenk et al, 2004. Int J Legal Med, 1 18 (1): 55-61) Together, the low percentages of stuttering alleles obtained for miniX favor the correct genotyping of the samples.
  • the PHR values define the balance in the heterozygous amplification.
  • miniX a lower average PHR is observed than that reported for other identification systems such as I-DNA-1 (Odriozola et al., 201 1 Int. J. Legal Med. 125 (5): 685-94), I- DNA-2 (Odriozola et al., 2012 Int. J. Legal Med. 126 (1): 167-72), geneRESMPX-3 (Schlenk et al, 2004. Int J Legal Med, 1 18 (1): 55- 61), MiniFiler TM PCR Amplification Kit (Mulero et al., 2008. J. Forensic Sci.
  • miniX shows a lower standard mean deviation than the one reported for Minificaler TM PCR Kit (Applied Biosystems, Foster City, CA) (12.02 vs. 33.47) (Mulero et al., 2008 J Forensic Sci. 53 (4): 838-52). It has not been possible to compare the percentage of stuttering alleles and PHR with reactions that include the STR loci of the reaction object of the invention, such as Decaplex (Gusmao et al., 2009 Int. J. Legal Med. 123 (3): 227 - 234) and Sextaplex (Casta ⁇ eda et al., 2012 J. Forensic Sci.
  • miniX The sensitivity of miniX has been compared with that reported for other STRs loci analysis systems and more specifically for those that include XS miniSTRs.
  • 5 (5) 415-421) require amounts greater than MiniX, at least 200 pg of template DNA to obtain complete profiles, and 100 pg for profiles with loss of 1-2 markers.
  • DNA extracts analyzed in forensic laboratories are often characterized by containing very small amounts of DNA, which makes it difficult to obtain genetic profiles of high discriminatory power after analysis using a multiplex STR system.
  • the size of the amplicons of a STR multiplex system is critical for obtaining genetic profiles from highly degraded biological samples (Budowle et al., 2009 Croat. Med. J. 50 (3): 207-17).
  • MiniX stands out for being the only system currently available that allows analyzing all the STRs DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS10079 loci, in a single multiplex reaction, also including amplified smaller than those available in the state of the art in 6 of the 8 STR loci included (Table 10). The exception is the DXS 10074 and DXS6801 locus that results in amplifications 27 and 37 bp greater than those obtained by the reactions developed by Becker et. al, 2008 Forensic. Sci. Int. Genet. 2 (1): 69-74 and Casta ⁇ eda et al., 2012 J.
  • miniX the smallest possible amplifications are produced for the DXS6809 locus (175-219 bp), having been designed in the area directly adjacent to the repetitive region of said locus. The rest of the STRs loci give rise to amplitudes less than 200 bp.
  • miniX represents the only alternative in miniSTR format for the amplification of the DXS6799, DXS 10075 and DXS 10079 loci. - -
  • miniX achieved greater performance than Decaplex in two of the 3 shared loci. While in the DXS7132 locus, both reactions obtained a yield of 88% by means of signals that exceeded 200 RFUs and Decaplex obtained an additional 4% by means of signals that did not reach said intensity. On the other hand, miniX exceeded the performance obtained through Sextaplex in the 6 STR loci shared by both reactions.
  • this validation includes both stuttering alleles, PHR and sensitivity assays, as well as comparative studies of design strategies and analysis of highly degraded DNA samples.
  • the results obtained demonstrate that the performance of the present invention in the analysis from highly degraded and / or scarce DNA samples of the DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS 10075 and DXS 10079 loci is superior to the prior art methods.

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Abstract

L'invention concerne une méthode pour obtenir le profil génétique d'un individu qui consiste à analyser dans un extrait d'ADN issu dudit individu, par une réaction d'amplification multiplex unique, de la combinaison de 8 loci STR du chromosome X constitués par DXS6799, DXS 10074, DXS6789, DXS6809, DXS7132, DXS6801, DXS10075 et DXS10079. L'invention concerne également un ensemble de paires d'amorces spécifiquement conçues pour l'amplification de chacun des 8 loci STR analysés dans une réaction multiplex unique et un kit qui comprend lesdites amorces pour la mise en oeuvre de la méthode.
PCT/ES2013/000170 2012-07-10 2013-07-09 Méthode pour l'obtention du profil génétique d'un individu par analyse de loci de chromosome x WO2014009578A1 (fr)

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CN110157812A (zh) * 2019-05-29 2019-08-23 苏州市公安局刑事科学技术研究所 一种同时检测常染色体和y染色体str基因座的复合扩增试剂盒
CN112195228A (zh) * 2020-09-28 2021-01-08 苏州阅微基因技术有限公司 X-str荧光扩增体系、试剂盒及应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110157812A (zh) * 2019-05-29 2019-08-23 苏州市公安局刑事科学技术研究所 一种同时检测常染色体和y染色体str基因座的复合扩增试剂盒
CN112195228A (zh) * 2020-09-28 2021-01-08 苏州阅微基因技术有限公司 X-str荧光扩增体系、试剂盒及应用
CN112195228B (zh) * 2020-09-28 2022-02-22 苏州阅微基因技术有限公司 X-str荧光扩增体系、试剂盒及应用

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