WO2015078997A1 - Method and kit for determining the sex and/or genetic footprint of a patient - Google Patents

Abstract

The present invention relates to a method for characterising a human or chimpanzee DNA sample, which includes a step of determining alleles of at least ten STR loci of the DNA sample, the loci being selected from the M2 group, wherein the M2 group consists of: Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533.

Description

 FIELD OF THE INVENTION:

 The present invention relates to a method and kit for characterizing a DNA sample of a human subject or chimpanzee to determine the sex and / or genetic imprint of that subject.

BACKGROUND OF THE INVENTION

 A genetic profile is the specific combination of several markers that can identify an individual through their DNA. There are essentially two main types of markers for establishing a genetic profile: Single Nucleotide Polymorphisms (SNPs), and Short Tandem Repeats (STRs).

An SNP corresponds to the change from one base to another at a location in the DNA sequence, for example a change from A to C. It thus comes in the form of 2 alleles. SNPs are characterized by a low mutation rate (2.1 x 10 ^"6 mutation / generation / locus) due to the low frequency of these mutations occur. These are excellent phylogenetic tools that tell us about the origin phylo- On the other hand, they are not discriminating enough to inform us about the relationships between closely related individuals, except when they are combined in large numbers (> 100 million to a million).

STRs are widely used in paternity research, rape and murder cases, and basic research to reconstruct the history of human settlement. They possess, a strong discriminating power, because of a high rate of mutation. These are tandem-repeated units (eg [CA] _e ) that mutate by gain or loss of a repetition, and come in the form of multiple states. Their mutation rate is higher or lower depending on their size, for example a CA _{6 will} mutate less rapidly than a CA These differences in mutation rates observed according to the reasons explains the broad spectrum of mutation of these markers (1.38 x 10 ^{~ 5} and 6.54 x 10 ^"2 mut / gen / locus) and their ability to discriminate more or less related individuals If the extreme mutability of some loci (> 10 ³ ) is very useful for discriminating genetically close individuals ( brothers, cousins), this one does not make it possible to obtain a robust information on the phylo-geographical origin of an individual (as it is possible to do it with SNPs) In addition, this hyper-mutability can induce a "mutational saturation" ie when a marker mutates too fast, the intermediate states of mutations are not always visible - this results in a poorly estimated mutation rate and derived, approximate and weak statistical indicators.

Ideally, a complete tool should be available, consisting of markers with a low mutation rate, able to give "phylogenetic" information on individuals, and markers with a medium or high mutation rate, able to discriminate even related individuals. Technically speaking, getting SNPs and STRs simply via the same technique, even in the context of next gen sequencing (NGS) sequencing, which rarely allows to extract very reliable STRs that remain relatively heavy to use routinely in a computer. Lambda laboratory. In forensics and basic research, there is a flagship product to establish a male genetic profile: the AmpRSTR® Yfiler © a 17-mark STR multiplexer distributed by Applied Biosystems (life technologies). However, this product does not respond well to the expectations of the various targeted clients because it contains few markers with a high mutation rate, essential in forensics to discriminate very close genetic profiles, as well as molecular genealogy and basic research. Moreover, among the few markers with a high mutation rate that it contains, two of them (DYS385a and b) are duplicated and therefore unusable without a thorough knowledge of the molecular mechanisms involved in their mutation. The markers included in this kit, with very variable rates and discriminating power, do not allow correct large-scale exploratory studies (weakly related: evolutionary genetics on the continental scale), nor correct studies on a fine scale (eg strong related in molecular genealogy or forensic medicine). Finally, the intrinsic characteristics of 5 of the remaining markers, do not allow to consider modeling studies to provide reliable statistics on mutation rates.

 Two similar tools have been developed by public laboratories: 20-plex (Butler et al., 2002, Forensic Sci. Int) and 14-plex (Parkin et al., 2006, Forensic Sci. Int): these 2 multiplexes although published 10 and 6 years ago respectively, did not impose themselves in the scientific community because using these tools in a laboratory would require a heavy investment of time, expertise and finances to set up the protocol in a laboratory. The investment is all the less justified since 70% of the markers are already included in the AmpFtSTR® Yfiler®; there is therefore no real added value to use them. In 2014 appeared on the market the PPY23 (Promega) which includes many markers already present in the AmpFtSTR® Yfiler®, and has the same limits. It should be noted that all these tools are human -specific, and that there is no equivalent of this type of tool for chimpanzees, whose management and conservation in parks and reserves, requires a sharp knowledge of relations genealogical relationships between individuals.

SUMMARY OF THE INVENTION:

The inventor has succeeded in designing a method for characterizing a DNA sample based on a set of genomic markers of the same type, which makes it possible to determine the high-resolution genetic fingerprint of a subject without presenting the aforementioned drawbacks. The set of markers according to the invention is usable not only on perfect DNAs, but also on medium-quality DNAs (old DNA or serum).

Indeed, these markers make it possible to work on fragments of relatively short size. Thus, these markers can be amplified with sizes always less than 350bp. In addition, the markers according to the invention are reliable / simple, ie they can be used without a thorough knowledge of molecular structures. Indeed, there is no duplication, false allele, and little complex structure, which should be considered in the use of rates or mutation mechanisms associated with these markers. A first aspect of the invention therefore relates to a method for characterizing a human or chimpanzee DNA sample comprising a step of:

 determination of the alleles of at least 10 STR loci of the DNA sample, the loci being chosen from the group M2, in which:

the group M2 consists of:

 Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533. The present invention also relates to a kit comprising means for determining the alleles of at least 10 STR loci, a sample of human or chimpanzee DNA, the loci being selected from the group M2, wherein:

the group M2 consists of:

 Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

 The term "allele" refers to one of the different forms of a DNA sequence occupying the same locus.

 The term "locus" refers to a specific position on a chromosome.

 The term "STR" or "Short Tandem Repeat" or "tandem repeat sequences" or microsatellite refers to all sequences between 2 and 7 nucleotides that are repeated in tandem.

Determination of the genetic profile of a subject

 The present invention relates to a method for characterizing a sample of human or chimpanzee DNA comprising a step of:

 determination of the alleles of at least 10 STR loci of the DNA sample, the loci being chosen from group M1 and / or group M2,

in which :

 the group M1 consists of:

DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538 and

 the group M2 consists of:

Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533. The present invention relates to a method for characterizing a sample of human or chimpanzee DNA comprising a step of: determination of the alleles of at least 10 STR loci of the DNA sample, the loci being chosen from the group M2,

in which :

 the group M2 consists of:

Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533.

 The method according to the invention makes it possible to determine the genetic profile of a subject.

 This method has the advantage of being both usable to determine the genetic profile of a man and that of a chimpanzee.

It makes it possible to discriminate the profiles of man and chimpanzee, the latter having neither the STRs DYS481 nor DYS533. The STRs Group M2 have high mutation rates between 3,32x10 ^{~ 03} mut / locus / generation and 1 45x10 ^"02 mut locus / generation (see Table 1). Due to their high mutation rate, these markers allow discrimination of relatively similar profiles, ie individuals with a close common ancestor in the past, ie with profiles that are found on a small geographic scale (regional, national), and / or genealogically bound.

 According to a preferred embodiment, the method according to the invention further comprises the step of determining the alleles of at least 10 STR loci of the DNA sample, the loci being chosen from the M1 group,

in which :

 Group 1 consists of:

DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538. STR M1 group have an intermediate transfer rates between 3,85x10 ^"04 mut / locus / generation and ^{3,21x10" 03} mut / locus / generation (see Table 1). Due to their lower mutation rate, compared to the existing markers included in the AmpFISTR®Yfiler, these markers allow discrimination of relatively divergent profiles, i.e. found on a large geographical scale (national, continental).

The determination of the alleles of the STR loci of the M1 group thus makes it possible to characterize individuals without direct family ties or kinships that are very far apart in time (evolutionary scale). The combination of the determination of alleles of the M2 group with that of the M1 group makes it possible to discriminate both individuals with very similar genetic profiles which is essential in forensic medicine but also to provide phylogenetic information about the phylogeographic origin of 'an individual.

In order to avoid cumbersome and costly analyzes, it is preferable to determine a limited number of STRs loci alleles. Thus, according to a preferred embodiment, the method according to the invention comprises the determination of the alleles of 10 to 50 STR loci, preferably the alleles of 10 to 40 STR loci, more preferably the alleles of 10 to 32 STR loci.

 Typically, the alleles of 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 loci can be determined.

 The choice of the number of loci depends on the desired use.

 If the method is used to discriminate very closely related individuals, 14 markers in M2 may be used to characterize a DNA sample from each of these individuals.

Whether the method is used to discriminate individuals from different continents or to study the phylogeographic origin of individual markers of 1 and 10 of M2 may be used to characterize a DNA sample from each of these individuals.

In a preferred embodiment, the alleles of the 32 loci are determined.

Similarly, the choice of STR loci depends on the desired use.

 In general, STR loci with a high mutation rate will be favored, which has a greater discriminating power.

 The mutation rates of the different loci are described in Table 1 below and ranked in ascending order of their mutation rate in humans. The molecular description of the STRs corresponding to these loci are given in Kayser et al. 2004 and Ballantyne et al. 2010.

Locus ID Details of

 Location of

 (Kayser location details Rate of

GBD ID Band Group

 et al. (retrotransposition, mutation

 chromosomal

 2004) median ...) median

 Bayesian

LINE1 L

 DYS502 1 Y3S60 Yq11.221 3,85E-04

 NLGN4Y mRNA

 DYS575 M1 Y4S49 Yp11.2 Alu L 3.91 E-04

DYS588 M1 Y5C23 YQ11.221 Alu R 3.92E-04

 Any

 DYS538 M1 Y4C32 Yq11.221 3.94E-04 retransposition

 DYS632 M1 BV005731 Yq11.221 Alu R 3.97E-04

DYS640 M1 Y4S38 YQ11.221 Alu L and LINEI R 3.98E-04

DYS485 M1 Y3S1 Yq 11.222 Alu R and LINEI L 4.04E-04

DYS577 M1 Y4S54 YQ11.221 Alu R 4.11 E-04

DYS461 /

 M1 - Yq 11.222 L1 The LINE family 9.89E-04 Y-GATA-A7.2

 Alu L, LINE ERV1

 DYS578 1 Y4S56 Yq 11.223 9.95E-04

 R

DYS638 1 Y4S26 Yq11.221 Alu R 1, 04E-03 Any

 (but close

 DYS511 M1 Y4C114 Yq11.221 1, 52E-03 from another

 simple repetition)

 DYS556 M1 Y4C75 YQ 11.223 LINE1 L and R 1, 59E-03

DYS565 M1 Y4S18 YQ11.221 Alu L 2.09E-03

 Any

 DYS587 M1 Y5C22 Yq11.221 2.62E-03 retransposition

 DYS508 M1 Y4C109 Yq11.221 None 3.03E-03

DYS517 1 Y4C126 Yq11.221 None 3.21 E-03

DYS643 M1 Y5C20 YQ11.221 LINE1 R 1, 50E-03

Y-GATA-A10 2 - Yq11.221 None 3,32E-03

DYS541 2 Y4C36 YQ11.221 LINE1 R 3.92E-03

DYS549 M2 Y4C52 YQ 11.222 LINE1 L and R 4,55E-03

DYS481 M2 Y3C59 Yp11.2 None 4,97E-03

 Any

 DYS533 M2 Y4C215 Yq11.221 (close to another 5.01 E-03

 Y-STR)

DYS444 M2 Y4C37 Yq11.221 Tigger2a DNA R5,45E-03

 Any

 (but close

 DYS510 M2 Y4C113 Yq11.221 5,99E-03 another simple

 repetition)

 DYS513 M2 Y4C116 Yq11.221 None 6.09E-03

DYS460 / (L1 R family

 2 - Yq 11.222 6.22E-03 Y-GATA-A7.1 LINE)

 DYS458 M2 - Yp11.2 Alu L and R 8,36E-03

DYS442 M2 - Y1 1q21 LINE1 L and R 9J8E-03

5'UTR of the gene

 DYS570 2 Y4S4 Yp11.2 TBL1 Y (coding 1, 24E-02 for a protein)

 DYS576 M2 Y4S5 Yp11.2 Alu L 1.43E-02

DYS612 M2 Y3C22 Yq11.221 None 1, 45E-02

Table 1

 In a second step, another criterion allowing the choice of the STR loci is the allelic range. These ranges are described in Tables 8A, 8B, 9A and 9B Plus the allelic ranges are large plus the STR loci potentially has significant discriminating power.

Finally, the 'pure' (eg (CA) n) patterns will be favored over the 'compound' motifs, ie the interrupted repeated motifs (eg CA3TGCT (CA) n). The markers according to the invention are male markers. Indeed, the loci are located on the Y chromosome, making it possible to generate a specifically male genetic profile. Obtaining an optimal male genetic profile is a real challenge in many fields not only in forensic medicine, since more than 97% of assaults are perpetrated by men but also in the context of paternity testing, genetic genealogy and history of settlement in order to trace the history of male lines of homo-sapiens. The method for characterizing a DNA sample can be performed on a sample whose sex has not been determined beforehand. In this case, since this method is based on male markers, it will only give results if the sample comes from a male. Nevertheless, the presence of included sexing markers will make it possible to say whether this non-amplification is due to the presence of a female individual (amplified X marker only), or of a highly degraded DNA (no marker is amplified).

 In a preferred embodiment, the DNA sample is a human DNA sample. The genetic profile of a human being, particularly a human being, is determined for example for forensic purposes as well as for research purposes for molecular genealogy and stand history studies.

 In another embodiment, the DNA sample is a chimpanzee DNA sample, particularly a male chimpanzee. For the chimpanzee, obtaining a genetic profile can be used in particular to determine the relationships of kinships of individuals in the wild and understand the social organization of this species. It can also optimize mating strategies, avoid inbreeding and ensure the good fate of the species when the chimpanzee is in semi-captivity or captivity. According to one embodiment, the method according to the invention comprises a step of providing a DNA sample.

The DNA sample can come from different sources. Examples of sources of DNA, in particular genomic DNA, are blood, sperm, hair, body hair, tissue, saliva, urine, faeces and mixtures of biological fluids. The DNA sample may be fresh, old, dry or partially degraded.

Methods for preparing a DNA sample to determine alleles of STR loci are known. For example, such methods are described in Patel, PI, et al. (1984) "Organization of the HPRT gene and related sequences in the human genome," Somat Cell Mol Genet 10: 483-493, and Gill, P., et al. (1985) "Forensic Application of DNA ^{'fingerprints',"} Nature 318: 577-579.

 From the DNA sample, the alleles of interest are determined.

Various methods of determining alleles of STR loci are known. Examples of such technologies include DNA sequencing or DNA amplification techniques such as PCR ("polymerase chain reaction" or "polymerization chain reaction"). These techniques can be used in combination with detection technologies such as electrophoresis, mass spectrometry, etc.

According to a preferred embodiment, the step of determining alleles comprises an amplification step such as PCR amplification. Loci alleles can be determined individually or simultaneously. Preferably, this amplification is performed simultaneously for several loci. This type of amplification is called multiplex amplification. Multiplex amplification is a process for simultaneously amplifying several loci in a single reaction. These multiplex amplifications have been widely described in the literature (Duchenne Muscular Dystrophy (Chamberlain, JS, et al., 1988), "Deletion Screening of the Duchenne Muscular Dystrophy Loci via Multiplex DNA Amplification," orral, N. and Estivill, X. ( 1992) "Multiplex PCR amplification of three STRs within the CFTR gene," Genomics 51: 1362- 1364); Kimpton, CP, et al. (1993) "Automated DNA profiling employing multiplex amplification of short tandem repeat loci," PCR Methods and Applications 3: 13-22 Hammond, Schumm, JW et al (1994) "Development of nonisotopic multiplex amplification sets for polymorphic analysis of STR loci," in "The Fourth International Symposium on Human Identification 1993," pp. 177-187 ).

 Multiplex amplifications are generally performed by PCR. This is called multiplex PCR.

The amplification step may generally comprise one or more of the following steps: denaturation of the DNA so that it is in single-stranded form,

 the addition of specific primer pairs for each of the STRs, one primer of each pair being substantially complementary to one part of the sequence in the sense strand and the other primer of each pair being substantially complementary to a different part of the same sequence on the complementary antisense strand,

 hybridization of the primer pairs to their complementary sequence,

 simultaneous elongation of the hybridized primers from the 3 'end of each primer to synthesize an extension product complementary to the hybridized strand with each primer, said elongation product, said elongation products, after separation of their complement serve as templates for the synthesis of one elongation product for the other primer of each pair,

 separation of the elongation products from the matrices to produce single-stranded molecules,

 the amplification of single-stranded molecules by repeating at least once the hybridization, elongation and separation steps.

 In one embodiment, the amplification step comprises the addition of at least 10 pairs of primers adapted to amplify the STR loci according to the invention.

 In one embodiment, the amplification step comprises adding 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 pairs of primers adapted to amplify the STR loci according to the invention.

 In one embodiment, the primer pairs have the nucleic sequences selected from the group consisting of:

 SEQ ID NO: 1 and SEQ ID NO: 2 for the DYS485 locus,

 SEQ ID NO: 3 and SEQ ID NO: 4 for the DYS588 locus,

 SEQ ID NO: 5 and SEQ ID NO: 6 for the DYS502 locus,

SEQ ID NO: 7 and SEQ ID NO: 8 for the DYS461 locus, SEQ ID NO: 9 and SEQ ID NO: 10 for the DYS638 locus,

 SEQ ID NO: 1 1 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO 13 and SEQ ID NO 14 for the DYS587 locus,

 SEQ ID NO 15 and SEQ ID NO 16 for the DYS575 locus,

 SEQ ID NO 17 and SEQ ID NO 18 for the DYS578 locus,

 SEQ ID NO 19 and SEQ ID NO 20 for the DYS632 locus,

 SEQ ID NO 21 and SEQ ID NO 22 for the DYS508 locus,

 SEQ ID NO 23 and SEQ ID NO 24 for the DYS640 locus,

 SEQ ID NO 25 and SEQ ID NO 26 for the DYS51 locus 1,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO 29 and SEQ ID NO 30 for the DYS556 locus,

 SEQ ID NO 31 and SEQ ID NO 32 for the DYS517 locus,

 SEQ ID NO 33 and SEQ ID NO 34 for the DYS565 locus,

 SEQ ID NO 35 and SEQ ID NO 36 for the DYS538 locus,

 SEQ ID NO 37 and SEQ ID NO 38 for the Y-GATA-A10 locus

 SEQ ID NO 39 and SEQ ID NO 40 for the DYS570 locus,

 SEQ ID NO 41 and SEQ ID NO 42 for the DYS549 locus,

 SEQ ID NO 43 and SEQ ID NO 44 for the DYS460 locus,

 SEQ ID NO 45 and SEQ ID NO 46 for the DYS442 locus

 SEQ ID NO 47 and SEQ ID NO 48 for the DYS510 locus,

 SEQ ID NO 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO 51 and SEQ ID NO 52 for the DYS576 locus,

 SEQ ID NO 53 and SEQ ID NO 54 for the DYS513 locus,

 SEQ ID NO 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO 57 and SEQ ID NO 58 for the DYS481 locus

 SEQ ID NO 59 and SEQ ID NO 60 for the DYS612 locus,

 SEQ ID NO 61 and SEQ ID NO 62 for the DYS444 locus and

 SEQ ID NO. 63 and SEQ ID NO. 64 for the DYS533 locus.

 The sequences of the primers of the various embodiments of the invention in which the DNA sample is a sample of human DNA or chimpanzee are given in Table 2 below.

Primer meaning Anti-sense primer

 SEQ SEQ ID marker

 Sequence (5'-3 ') Sequence (5'-3') ID NO NO

 catataacaaaattgaatgtgtactc

 DYS485 1 2 agcctgggtgacaagagttatac

 vs

 DYS588 3 gaatgcagaaccctcaagga 4 agcctgggtgacagaaacac

DYS502 5 cag caag ccaccataccata 6 Tgtgcttttggagtttggag DYS461 /

 aatacataataaatgataggcaga

Y-GATA- 7 8 gagagctgaataagttgtatcaggtaa gga

A7.2

 DYS638 9 ttctaatttcagtgctttcaattttc 10 Aggtgggtcatgaggtcagt

 DYS643 1 1 aag ccatg cctggttaaactac 12 accaaacaccacccattcc

 aaagtctgacaatgagaagggtttctaagtt

DYS587 13 cttctttggaaagtagcatttcat 14

 ACGG

 DYS575 15 cagaggttccagtaagcttagatca 16 Cattgatgggctttaggttga

 DYS578 17 gaggcggaactttcagtgag 18 cagaagtcccctgtgtttcaa

 DYS632 19 cacagtttcagtcttgcatgg 20 tctgggcaacagaaggagac

 DYS508 21 acaatgg caatcccaaattc 22 gaacaaataaggtgggatggat

 DYS640 23 ggaaaaaccatgagatcctgtc 24 aag cccgttcatattttaaagac

 DYS511 25 tggggtggatgtgtaggtaga 26 Tctggttgtgccttagatttga

 DYS577 27 tttttctacgtgtgtatccactaacc 28 Gtgtccccagccctgtta

 DYS556 29 tcaccaatgacattttacagca 30 ttggttagtgtaatgcatccag

 DYS517 31 aactgaccagcaaaaatgttaaa 32 tgtctgagacctacaagattgc

 DYS565 33 ccaggaagcagtgttgcat 34 Gcagttctctgcctgtatgg

 DYS538 35 ttggggaaaacagatggtgt 36 ccaaatacccatcataggaagaa

 Are GATA-

37 cctg ccatctctatttatcttg catata 38 ataaatggagatagtgggtggatt

A10

 DYS570 39 tgtgacatcaaggttatgaaacg 40 ggtgaaaattattcagcatagtcaag gaaaagaaaagtgtaagccaaac

 DYS549 41 42 tttggtggcataagtggtaatg

 vs

 DYS460 43 atcctctg cctatcatttattatgtat 44 gaataccagaggaatctgacacc

 DYS442 45 tgcaaaatcacggaaccaa 46 Caag cca ctg caaatg tca

 DYS510 47 tttttcctcccttaccacaga 48 tctggagaagacagaacttgtca

 DYS541 49 catcattaattctatctgttcatccat 50 tggataaagaacacctttaagaagc

DYS576 51 ccaag caacatag caag acct 52 Aagcgtatttgtcttggctttt

 tgttgtaaaaatgactactgtggtat

 DYS513 53 54 ccacatcag cactattacttaactca g

 DYS458 55 tgggtggtggaggttactgt 56 cccaaagttctggcattacaa

 DYS481 57 aggaatgtggctaacgctgt 58 accagaaggttgcaagactca

 DYS612 59 cccccatg ccag taagaata 60 tgagggaaggcaaaagaaaa

 DYS444 61 catagaatgaaaggtgtgaacca 62 Tgccattcaaactcacgttg

 DYS533 63 attcatctaacatctttgtcatctacc 64 Ttaacttg cctttttg catcc

Table 2 Note, however, that the DYS481 locus is absent in the chimpanzee, and that the DYS533 locus, although theoretically present on the assembled genome of the chimpanzee (XXX), could never be amplified in the test phase - it is therefore considered unamplified in M2. Therefore, in one embodiment, the DNA sample is a chimpanzee DNA sample and the primer pairs have the nucleic sequences selected from the group consisting of:

 SEQ ID NO 1 and SEQ ID NO: 2 for the DYS485 locus,

 SEQ ID NO 3 and SEQ ID NO: 4 for the DYS588 locus,

 SEQ ID NO 5 and SEQ ID NO: 6 for the DYS502 locus,

 SEQ ID NO 7 and SEQ ID NO: 8 for the DYS461 locus,

 SEQ ID NO 9 and SEQ ID NO: 10 for the DYS638 locus,

 SEQ ID NO 11 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO 13 and SEQ ID NO: 14 for the DYS587 locus,

 SEQ ID NO 15 and SEQ ID NO: 16 for the DYS575 locus,

 SEQ ID NO 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO 19 and SEQ ID NO: 20 for the DYS632 locus,

 SEQ ID NO: 21 and SEQ ID NO: 22 for the DYS508 locus,

 SEQ ID NO: 23 and SEQ ID NO: 24 for the DYS640 locus,

 SEQ ID NO: 25 and SEQ ID NO: 26 for the DYS511 locus,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO: 29 and SEQ ID NO: 30 for the DYS556 locus,

 SEQ ID NO: 31 and SEQ ID NO: 32 for the DYS517 locus,

 SEQ ID NO: 33 and SEQ ID NO: 34 for the DYS565 locus,

 SEQ ID NO: 35 and SEQ ID NO: 36 for the DYS538 locus,

 SEQ ID NO: 37 and SEQ ID NO: 38 for the Y-GATA-A10 locus

 SEQ ID NO: 39 and SEQ ID NO: 40 for the DYS570 locus,

 SEQ ID NO: 41 and SEQ ID NO: 42 for the DYS549 locus,

 SEQ ID NO: 43 and SEQ ID NO: 44 for the DYS460 locus,

 SEQ ID NO: 45 and SEQ ID NO: 46 for the DYS442 locus

 SEQ ID NO: 47 and SEQ ID NO: 48 for the DYS510 locus,

 SEQ ID NO: 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO: 51 and SEQ ID NO: 52 for the DYS576 locus,

 SEQ ID NO: 53 and SEQ ID NO: 54 for the DYS513 locus,

 SEQ ID NO: 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO: 59 and SEQ ID NO: 60 for the DYS612 locus and

 SEQ ID NO: 61 and SEQ ID NO: 62 for the DYS444 locus.

In some embodiments, the PCR extension products can be detected by fluorescent markers conjugated to the PCR amplification primers (see for example WO2009 / 059049). PCR products can also be detected by other techniques such as, for example, the staining of amplification products such as silver staining, radioisotopes, by chemiluminescence.

 Examples of fluorescent markers are FAM (5-carboxyfluorescein, fluorescent in blue), VIC® (fluorescent in green, available from Life Techologies), NED ™ (fluorescent in yolk available from Life Techologies) and PET® ( fluorescent in the red available from Life Techologies), TET (tetrachlorofluorescein, fluorescent in the green), and HEX (6-carboxy 2,4,7,4,7-hexachlorofluorescein, fluorescent in the yolk).

In one embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of at least 15 STR loci selected from the M1 group.

M1 group STRs have a low to intermediate mutation rate. Their mutation rate is between 3,85x10 ^{~ 04} mut / locus / generation and 3,21x10 ^"03 mut / locus / generation (see Table 1) Due to their relatively low mutation rate, compared to the main markers These markers, like those of the AmpFistr®Yfiler, allow for relatively divergent profile discrimination, ie that can be found on a large geographical scale, for example at the level of countries or continents.

 In one embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of 15, 16, 17 or 18 STR loci selected from the M1 group. In a preferred embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of the 18 STR loci of the M1 group.

 In one embodiment, the method for characterizing a sample comprises the step of determining the alleles of at least 11 STR loci selected in the M2 group.

STRs in the M2 group have a high mutation rate. Their mutation rate is between 3.32x10 ^"03 mut / locus / generation and 1.45x10 ^{" 02} mut / locus / generation (see Table 1). Due to their high mutation rate, these markers allow discrimination of relatively similar profiles, i.e. that can be found on a small geographical scale, for example on the scale of a region.

 In one embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of 11, 12, 13 or 14 STR loci selected in the M2 group. In another preferred embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of the 14 STR group M2 loci.

 In another embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of the 14 STR loci in the M2 group only. In this embodiment, the method does not include determining alleles of STR loci other than those of the M2 group.

 In one embodiment, the method for characterizing a DNA sample comprises a step of determining alleles:

 at least 10 STR loci chosen in group 1,

and or

at least 10 STR loci chosen in the group M2. In a preferred embodiment, the loci whose alleles are determined are at least DYS485, DYS588, DYS461, DYS638, DYS643, DYS587, DYS517, DYS508, DYS511 and DYS565 for the M1 group and / or at least DYS570, DYS510, DYS541. , DYS576, DYS458, DYS481, DYS612, DYS460, DYS442, DYS513 and DYS444 for the M2 group.

More preferably, the loci whose alleles are determined are at least DYS485, DYS588, DYS461, DYS638, DYS643, DYS587, DYS517, DYS508, DYS511, DYS565, DYS578, DYS556 and DYS538 for the M1 group and / or at least DYS570, DYS510. , DYS541, DYS576, DYS458, DYS481, DYS612, DYS460, DYS442, DYS513 and DYS444 for the M2 group.

In one embodiment, the method for characterizing a DNA sample comprises a step of determining alleles:

 from 10, 1 1, 12, 13, 14, 15, 16, 17 or 18 STR loci selected from the M1 group,

and or

 of 10, 1 1, 12, 13 or 14 STR loci selected in group M2.

 In a preferred embodiment, the method for characterizing a DNA sample comprises a step of determining the alleles of the 18 M1 STR loci and the 14 M1 STR loci.

 In another embodiment, the method for characterizing a DNA sample comprises the step of determining the alleles of the 18 M1 STR loci and the 14 M1 STR loci only. In this embodiment, the method does not include determining alleles of STR loci other than those of groups M1 and M2.

 The combination of the STR loci of the two sets M1 and M2 allows to obtain very informative genetic profiles, providing information as well on the links existing between individuals little as strongly related.

 It is also interesting to use all the loci of groups M1 and 2 in populations with low genetic diversity.

 For example, in chimpanzees, this species has undergone significant reductions in numbers, and its genetic diversity has greatly weakened. Therefore, the use of all markers will allow very useful maximum discrimination on this type of population with low genetic diversity.

The method for characterizing a DNA sample can be performed on a sample whose sex has not been determined beforehand. In this case, since this method is based on male markers, it will only give results if the sample comes from a male.

 Preferably, the method for characterizing a DNA sample is used on a sample which is known to be from a male.

 According to one embodiment, the method for characterizing a DNA sample may further comprise a step of determining the sex of the subject from which the DNA sample originates.

Methods for determining the sex of a subject are known. Most of these methods rely on amplification of the amelogenin gene on X and Y (see Sullivan et al., 1993, Ensminger and Hoffman 2002). Such methods are commercially available through kits such as the PowerPlex16 (Promega) and the AmpF / STR Identifiler (Life Technologies). Rarer, there are also sex determination methods based on the amplification of two distinct loci on the sex chromosomes (ie DXS8378, DXS6803 on the X chromosome and SRY on the Y chromosome).

 According to one embodiment, the step of determining the sex of a DNA sample which comprises a step of amplifying the SRY, UTY and UTX genes of the DNA sample. This method for determining the sex of a subject makes it possible, in addition to identifying the sex, to indirectly evaluate the degradation profile of the male DNA sample studied.

 Indeed, the 3 markers having sizes of 81 (SRY), 91 (UTY) and 120 (UTX) bp when the sample comes from a man or a chimpanzee, the decreasing amplification of these 3 markers informs us on the degree of fragmentation of the sample. This is particularly interesting when using this method of sex determination before determining the genetic profile of a subject further.

 Thus, by proceeding in advance with this step, it is possible to quickly evaluate whether the sample is suitable for being analyzed or not.

 In addition, sometimes the sexing of a sample prior to the determination of a genetic profile of an individual fails. Thus, some samples identified as female, are, in fact, male samples for which sexing has failed. Combining the determination of an individual's genetic profile with a sexing tool validates the sexing done prior to the determination of the genetic fingerprint.

 In one embodiment, the step of amplifying the SRY, UTY and UTX genes comprises adding primer pairs having the nucleic sequences:

 SEQ ID NO: 65 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

 and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

 Table 3

 Kit to characterize a DNA sample

The present invention also relates to a kit comprising means for determining the alleles of at least 10 STR loci of a human or chimpanzee DNA sample, the loci being selected from the M1 group and / or the M2 group, wherein : the group M1 consists of:

DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538 and

the group M2 consists of:

 Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533.

 The present invention also relates to a kit comprising means for determining the alleles of at least 10 STR loci of a human or chimpanzee DNA sample, the loci being selected from the group M2, wherein:

the group M2 consists of:

 Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533.

 The kit may include means for determining alleles of STR loci other than those in the M2 group.

 For reasons of cost and ease of use, it is preferable that the alleles to be determined be limited.

 Thus, the kit may include means for determining alleles of 10 to 50 STR loci, preferably alleles of 10 to 40 STR loci, more preferably alleles of 10 to 32 STR loci.

 Typically, the kit comprises the means for determining the alleles of 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31 or 32 STR loci.

In a preferred embodiment, the kit comprises at least 10 pairs of primers adapted to amplify the STR loci, the loci being selected from Group 1 and / or the M2 group.

Typically, the kit comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 pairs of primers.

 In one embodiment, the DNA sample is a sample of human or chimpanzee DNA and the at least 10 primer pairs have the nucleic sequences selected from the group consisting of:

 SEQ ID NO: 1 and SEQ ID NO: 2 for the DYS485 locus,

 SEQ ID NO: 3 and SEQ ID NO: 4 for the DYS588 locus,

 SEQ ID NO: 5 and SEQ ID NO: 6 for the DYS502 locus,

 SEQ ID NO: 7 and SEQ ID NO: 8 for the DYS461 locus,

 SEQ ID NO: 9 and SEQ ID NO: 10 for the DYS638 locus,

 SEQ ID NO: 1 1 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO: 13 and SEQ ID NO: 14 for the DYS587 locus,

 SEQ ID NO: 15 and SEQ ID NO: 16 for the DYS575 locus,

 SEQ ID NO: 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO: 19 and SEQ ID NO: 20 for the DYS632 locus,

 SEQ ID NO: 21 and SEQ ID NO: 22 for the DYS508 locus,

SEQ ID NO: 23 and SEQ ID NO: 24 for the DYS640 locus, SEQ ID NO: 25 and SEQ ID NO:: 26 for the DYS511 locus,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO: 29 and SEQ ID NO:: 30 for the DYS556 locus,

 SEQ ID NO: 31 and SEQ ID NO:: 32 for the DYS517 locus,

 SEQ ID NO: 33 and SEQ ID NO:: 34 for the DYS565 locus,

 SEQ ID NO: 35 and SEQ ID NO:: 36 for the DYS538 locus,

 SEQ ID NO: 37 and SEQ ID NO:: 38 for the Y-GATA-A10 locus

 SEQ ID NO: 39 and SEQ ID NO:: 40 for the DYS570 locus,

 SEQ ID NO: 41 and SEQ ID NO:: 42 for the DYS549 locus,

 SEQ ID NO: 43 and SEQ ID NO:: 44 for the DYS460 locus,

 SEQ ID NO: 45 and SEQ ID NO:: 46 for the DYS442 locus

 SEQ ID NO: 47 and SEQ ID NO:: 48 for the DYS510 locus,

 SEQ ID NO: 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO: 51 and SEQ ID NO:: 52 for the DYS576 locus,

 SEQ ID NO: 53 and SEQ ID NO:: 54 for the DYS513 locus,

 SEQ ID NO: 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO: 57 and SEQ ID NO:: 58 for the DYS481 locus

 SEQ ID NO: 59 and SEQ ID NO:: 60 for the DYS612 locus,

 SEQ ID NO: 61 and SEQ ID NO:: 62 for the DYS444 locus and

 SEQ ID NO: 63 and SEQ ID NO:: 64 for the DYS533 locus.

 In one embodiment, the DNA sample is a chimpanzee DNA sample and the at least 10 primer pairs have the nucleic sequences selected from the group consisting of:

 SEQ ID NO: 1 and SEQ ID NO 2 for the DYS485 locus,

 SEQ ID NO: 3 and SEQ ID NO 4 for the DYS588 locus,

 SEQ ID NO: 5 and SEQ ID NO 6 for the DYS502 locus,

 SEQ ID NO: 7 and SEQ ID NO 8 for the DYS461 locus,

 SEQ ID NO: 9 and SEQ ID NO 10 for the DYS638 locus,

 SEQ ID NO: 1 1 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO: 13 and SEQ ID NO: 14 for the DYS587 locus,

 SEQ ID NO: 15 and SEQ ID NO: 16 for the DYS575 locus,

 SEQ ID NO: 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO: 19 and SEQ ID NO: 20 for the DYS632 locus,

 SEQ ID NO: 21 and SEQ ID NO: 22 for the DYS508 locus,

 SEQ ID NO: 23 and SEQ ID NO: 24 for the DYS640 locus,

 SEQ ID NO: 25 and SEQ ID NO: 26 for the DYS511 locus,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO: 29 and SEQ ID NO: 30 for the DYS556 locus,

 SEQ ID NO: 31 and SEQ ID NO: 32 for the DYS517 locus,

 SEQ ID NO: 33 and SEQ ID NO: 34 for the DYS565 locus,

SEQ ID NO: 35 and SEQ ID NO: 36 for the DYS538 locus, SEQ ID NO:: 37 and SEQ ID NO: 38 for the Y-GATA-A10 locus

 SEQ ID NO:: 39 and SEQ ID NO: 40 for the DYS570 locus,

 SEQ ID NO:: 41 and SEQ ID NO: 42 for the DYS549 locus,

 SEQ ID NO:: 43 and SEQ ID NO: 44 for the DYS460 locus,

 SEQ ID NO:: 45 and SEQ ID NO: 46 for the DYS442 locus

 SEQ ID NO:: 47 and SEQ ID NO: 48 for the DYS510 locus,

 SEQ ID NO:: 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO:: 51 and SEQ ID NO: 52 for the DYS576 locus,

 SEQ ID NO:: 53 and SEQ ID NO: 54 for the DYS513 locus,

 SEQ ID NO:: 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO:: 59 and SEQ ID NO: 60 for the DYS612 locus and

 SEQ ID NO:: 61 and SEQ ID NO: 62 for the DYS444 locus.

 In a preferred embodiment, the DNA sample is a sample of human or chimpanzee DNA and the kit comprises primer pairs having nucleic sequences selected from the group consisting of:

 SEQ ID NO 37 and SEQ ID NO

 SEQ ID NO 39 and SEQ ID NO

 SEQ ID NO 41 and SEQ ID NO

 SEQ ID NO 43 and SEQ ID NO

 SEQ ID NO 45 and SEQ ID NO

 SEQ ID NO 47 and SEQ ID NO

 SEQ ID NO 49 and SEQ ID NO

 SEQ ID NO 51 and SEQ ID NO

 SEQ ID NO 53 and SEQ ID NO

 SEQ ID NO 55 and SEQ ID NO

 SEQ ID NO 57 and SEQ ID NO

 SEQ ID NO 59 and SEQ ID NO

 SEQ ID NO 61 and SEQ ID NO

 SEQ ID NO 63 and SEQ ID NO

 In a preferred embodiment, the

the alleles of at least 10 STR loci selected from group M1,

in which :

 the group M1 consists of:

DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538.

 According to this preferred embodiment, the kit may comprise at least 10 pairs of primers adapted to amplify the STR loci and

wherein the primer pairs have nucleic sequences selected from the group consisting of:

 SEQ ID NO: 1 and SEQ ID NO: 2 for the DYS485 locus,

SEQ ID NO: 3 and SEQ ID NO: 4 for the DYS588 locus, SEQ ID NO 5 and SEQ ID NO: 6 for the DYS502 locus,

 SEQ ID NO 7 and SEQ ID NO: 8 for the DYS461 locus,

 SEQ ID NO 9 and SEQ ID NO: 10 for the DYS638 locus,

 SEQ ID NO 11 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO 13 and SEQ ID NO: 14 for the DYS587 locus,

 SEQ ID NO 15 and SEQ ID NO: 16 for the DYS575 locus,

 SEQ ID NO 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO 19 and SEQ ID NO: 20 for the DYS632 locus,

 SEQ ID NO 21 and SEQ ID NO: 22 for the DYS508 locus,

 SEQ ID NO 23 and SEQ ID NO: 24 for the DYS640 locus,

 SEQ ID NO 25 and SEQ ID NO: 26 for the DYS51 locus 1,

 SEQ ID NO: 27 and SEQ ID NO 28 for the DYS577 locus,

 SEQ ID NO 29 and SEQ ID NO: 30 for the DYS556 locus,

 SEQ ID NO 31 and SEQ ID NO: 32 for the DYS517 locus,

 SEQ ID NO 33 and SEQ ID NO: 34 for the DYS565 locus,

 SEQ ID NO 35 and SEQ ID NO: 36 for the DYS538 locus,

SECTION M1 In one embodiment, the kit comprises means for determining the alleles of at least 15 STR loci selected from the M1 group.

 In this embodiment, the kit comprises means for determining the alleles of 15, 16, 17 or 18 loci selected from the M1 group.

 In a preferred embodiment, the kit comprises means for determining the alleles of the 18 loci of the M 1 group.

In a preferred embodiment, the kit comprises means for determining the alleles of at least the DYS485, DYS588, DYS461, DYS638, DYS643, DYS587, DYS517, DYS508, DYS51 1 and DYS565 loci, more preferably at least DYS485 loci, DYS588, DYS461, DYS638, DYS643, DYS587, DYS517, DYS508, DYS511, DYS565, DYS578, DYS556 and DYS538.

 In a preferred embodiment, the kit comprises primer pairs having the nucleic sequences:

 SEQ ID NO: 1 and SEQ ID NO: 2 for the DYS485 locus,

 SEQ ID NO: 3 and SEQ ID NO: 4 for the DYS588 locus,

 SEQ ID NO: 5 and SEQ ID NO: 6 for the DYS502 locus,

 SEQ ID NO: 7 and SEQ ID NO: 8 for the DYS461 locus,

 SEQ ID NO: 9 and SEQ ID NO: 10 for the DYS638 locus,

 SEQ ID NO: 11 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO: 13 and SEQ ID NO: 14 for the DYS587 locus,

SEQ ID NO: 15 and SEQ ID NO: 16 for the DYS575 locus, SEQ ID NO 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO 19 and SEQ ID NO: 20 for the DYS632 locus,

 SEQ ID NO 21 and SEQ ID NO: 22 for the DYS508 locus,

 SEQ ID NO 23 and SEQ ID NO: 24 for the DYS640 locus,

 SEQ ID NO 25 and SEQ ID NO: 26 for the DYS51 locus 1,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO 29 and SEQ ID NO: 30 for the DYS556 locus,

 SEQ ID NO 31 and SEQ ID NO: 32 for the DYS517 locus,

 SEQ ID NO 33 and SEQ ID NO: 34 for the DYS565 locus and

 SEQ ID NO: 35 and SEQ ID NO: 36 for the DYS538 locus.

 These pairs of primers correspond to the primer pairs adapted to amplify the STR loci of the M1 group. They can be used for both a human DNA sample and a chimpanzee DNA sample. M1 group STRs have a low to intermediate mutation rate. These markers allow discrimination of relatively divergent profiles.

SECTION M2

 In one embodiment, the kit comprises means for determining the alleles of at least 11 STR loci selected in the M2 group.

 In this embodiment, the kit comprises means for determining alleles of 1, 12, 13 or 14 loci selected from group 2.

 In another preferred embodiment, the kit comprises means for determining the alleles of the 14 loci of the M2 group.

 In another embodiment, the kit comprises means for determining the alleles of the 14 loci of the M2 group only. In this embodiment, the method does not include determining alleles of STR loci other than those of group 2.

 In a preferred embodiment, the kit comprises means for determining the alleles of at least the DYS570, DYS510, DYS541, DYS576, DYS458, DYS481, DYS612, DYS460, DYS442, DYS513 and DYS444 loci.

 In a preferred embodiment, the DNA sample is a sample of human or chimpanzee DNA and the kit comprises primer pairs having the nucleic sequences:

 SEQ ID NO 37 and SEQ ID NO 38 for the Y-GATA-A10 locus

 SEQ ID NO 39 and SEQ ID NO 40 for the DYS570 locus,

 SEQ ID NO 41 and SEQ ID NO 42 for the DYS549 locus,

 SEQ ID NO 43 and SEQ ID NO 44 for the DYS460 locus,

 SEQ ID NO 45 and SEQ ID NO 46 for the DYS442 locus

 SEQ ID NO 47 and SEQ ID NO 48 for the DYS510 locus,

 SEQ ID NO 49 and SEQ ID NO 50 for the DYS541 locus,

 SEQ ID NO 51 and SEQ ID NO 52 for the DYS576 locus,

 SEQ ID NO 53 and SEQ ID NO 54 for the DYS513 locus,

SEQ ID NO 55 and SEQ ID NO 56 for the DYS458 locus, SEQ ID NO 57 and SEQ ID NO: 58 for the DYS481 locus

 SEQ ID NO 59 and SEQ ID NO: 60 for the DYS612 locus,

 SEQ ID NO 61 and SEQ ID NO: 62 for the DYS444 locus and

 SEQ ID NO. 63 and SEQ ID NO: 64 for the DYS533 locus.

STRs in the M2 group have a high mutation rate. These markers allow discrimination of profiles on a smaller scale, for example at the scale of a region.

In another embodiment, the DNA sample is a chimpanzee DNA sample and the kit comprises the primer pairs having the nucleic sequences:

 SEQ ID NO 37 and SEQ ID NO 38 for the Y-GATA-A10 locus

 SEQ ID NO 39 and SEQ ID NO 40 for the DYS570 locus,

 SEQ ID NO 41 and SEQ ID NO 42 for the DYS549 locus,

 SEQ ID NO 43 and SEQ ID NO 44 for the DYS460 locus,

 SEQ ID NO 45 and SEQ ID NO 46 for the DYS442 locus

 SEQ ID NO 47 and SEQ ID NO 48 for the DYS510 locus,

 SEQ ID NO 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO 51 and SEQ ID NO 52 for the DYS576 locus,

 SEQ ID NO 53 and SEQ ID NO 54 for the DYS513 locus,

 SEQ ID NO 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO 59 and SEQ ID NO 60 for the DYS612 locus and

 SEQ ID NO 61 and SEQ ID NO 62 for the DYS444 locus.

SECTION M1 and M2

 In one embodiment, the kit includes means for determining the alleles:

 at least 10 STR loci chosen in group M1

and

 at least 10 STR loci chosen in group M2.

 In this embodiment, the kit comprises means for determining the alleles:

 - 10, 1 1, 12, 13, 14, 15, 16, 17 or 18 STR loci chosen in group 1

and

 - 10, 1 1, 12, 13 or 14 STR loci selected in the group M2.

 In a preferred embodiment, the kit comprises means for determining the 18 STR loci of the M1 group and the 14 STR loci of the M2 group.

 In a preferred embodiment, the DNA sample is a sample of human or chimpanzee DNA and the kit comprises primer pairs having the nucleic sequences:

 SEQ ID NO I and SEQ ID NO: 2 for the DYS485 locus,

 SEQ ID NO 3 and SEQ ID NO: 4 for the DYS588 locus,

 SEQ ID NO 5 and SEQ ID NO: 6 for the DYS502 locus,

 SEQ ID NO 7 and SEQ ID NO: 8 for the DYS461 locus,

 SEQ ID NO 9 and SEQ ID NO: 10 for the DYS638 locus,

SEQ ID NO II and SEQ ID NO: 12 for the DYS643 locus, SEQ ID NO 13 and SEQ ID NO: 14 for the ocus DYS587,

 SEQ ID NO 15 and SEQ ID NO: 16 for the ocus DYS575,

 SEQ ID NO 17 and SEQ ID NO: 18 for the ocus DYS578,

 SEQ ID NO 19 and SEQ ID NO: 20 for the ocus DYS632,

 SEQ ID NO 21 and SEQ ID NO: 22 for the ocus DYS508,

 SEQ ID NO 23 and SEQ ID NO: 24 for the ocus DYS640,

 SEQ ID NO 25 and SEQ ID NO: 26 for the ocus DYS51 1,

 SEQ ID NO 27 and SEQ ID NO 28 for the DYS577 locus,

 SEQ ID NO 29 and SEQ ID NO 30 for the DYS556 locus,

 SEQ ID NO 31 and SEQ ID NO 32 for the DYS517 locus,

 SEQ ID NO 33 and SEQ ID NO 34 for the ocus DYS565,

 SEQ ID NO 35 and SEQ ID NO 36 for the DYS538 locus,

SEQ ID NO 37 and SEQ ID NO 38 for the ocus Y-GATA-A10

 SEQ ID NO 39 and SEQ ID NO 40 for the DYS570 locus,

 SEQ ID NO 41 and SEQ ID NO 42 for the DYS549 locus,

 SEQ ID NO 43 and SEQ ID NO 44 for the DYS460 locus,

 SEQ ID NO 45 and SEQ ID NO 46 for the ocus DYS442

 SEQ ID NO 47 and SEQ ID NO 48 for the DYS510 locus,

SEQ ID NO 49 and SEQ ID NO: 50 poui ^• the DYS541 locus,

 SEQ ID NO 51 and SEQ ID NO 52 for the DYS576 locus,

 SEQ ID NO 53 and SEQ ID NO 54 for the ocus DYS513,

 SEQ ID NO 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO. 57 and SEQ ID NO. 58 for the ocus DYS481

 SEQ ID NO 59 and SEQ ID NO 60 i for the DYS612 locus,

 SEQ ID NO 61 and SEQ ID NO 62! for the ocus DYS444 and

 SEQ ID NO 63 and SEQ ID NO 64 ■ for the ocus DYS533.

In another embodiment, the DNA sample is a chimpanzee DNA sample and the kit comprises the primer pairs having the nucleic sequences:

 SEQ ID NO: 1 and SEQ ID NO 2 for the DYS485 locus,

 SEQ ID NO: 3 and SEQ ID NO 4 for the DYS588 locus,

 SEQ ID NO: 5 and SEQ ID NO 6 for the DYS502 locus,

 SEQ ID NO: 7 and SEQ ID NO 8 for the DYS461 locus,

 SEQ ID NO: 9 and SEQ ID NO 10 for the DYS638 locus,

 SEQ ID NO: 1 1 and SEQ ID NO: 12 for the DYS643 locus,

 SEQ ID NO: 13 and SEQ ID NO: 14 for the DYS587 locus,

 SEQ ID NO: 15 and SEQ ID NO: 16 for the DYS575 locus,

 SEQ ID NO: 17 and SEQ ID NO: 18 for the DYS578 locus,

 SEQ ID NO: 19 and SEQ ID NO: 20 for the DYS632 locus,

SEQ ID NO: 21 and SEQ ID NO: 22 for the DYS508 locus, SEQ ID NO: 23 and SEQ ID NO: 24 for the DYS640 locus,

 SEQ ID NO: 25 and SEQ ID NO: 26 for the DYS511 locus,

 SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus,

 SEQ ID NO: 29 and SEQ ID NO: 30 for the DYS556 locus,

 SEQ ID NO: 31 and SEQ ID NO: 32 for the DYS517 locus,

 SEQ ID NO: 33 and SEQ ID NO: 34 for the DYS565 locus,

 SEQ ID NO: 35 and SEQ ID NO: 36 for the DYS538 locus,

SEQ ID NO 37 and SEQ ID NO 38 for the Y-GATA-A10 locus

 SEQ ID NO 39 and SEQ ID NO 40 for the DYS570 locus,

 SEQ ID NO 41 and SEQ ID NO 42 for the DYS549 locus,

 SEQ ID NO 43 and SEQ ID NO 44 for the DYS460 locus,

 SEQ ID NO 45 and SEQ ID NO 46 for the DYS442 locus

 SEQ ID NO 47 and SEQ ID NO 48 for the DYS510 locus,

 SEQ ID NO 49 and SEQ ID NO: 50 for the DYS541 locus,

 SEQ ID NO 51 and SEQ ID NO 52 for the DYS576 locus,

 SEQ ID NO 53 and SEQ ID NO 54 for the DYS513 locus,

 SEQ ID NO 55 and SEQ ID NO: 56 for the DYS458 locus,

 SEQ ID NO 59 and SEQ ID NO 60 for the DYS612 locus and

 SEQ ID NO 61 and SEQ ID NO 62 for the DYS444 locus.

In one embodiment, the kit further comprises gender determination means, wherein the gender determination means comprises SRY, UTY and UTX gene amplification means.

In one embodiment, the SRY, UTY and UTX gene amplification means comprise the primer pairs having the nucleic sequences:

SEQ ID NO: 65 and SEQ ID NO: 66 for SRY,

SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

The present invention also relates to the use of a kit as defined above for carrying out the method according to the invention.

Sexaqe tool:

The present invention also relates to a method for determining the sex of a DNA sample.

¹ molecular tool developed in humans for the sex identification is the test of amelogenin. This test is based on the use of a single pair of PCR primers capable of co-amplify DNA fragments on both amelogenin genes of chromosomes X and Y, which in humans differ by 6 bp in intron 1. This size difference is usually resolved by capillary electrophoresis, revealing a single peak for females (XX) and a double peak for males (XY). This method is today the standard used for sex determination in forensics and population genetics; this test is included in 2 kits including PowerPlex16 (Promega) and AmpF / STR Indentifiler (Life Technologies). Nevertheless, despite its long-term use, this sex typing trial is not entirely reliable. Many studies have reported an erroneous sex determination because the AMELY locus could not be amplified. Two causes are involved: a mutation in the binding site of the primer and / or the deletion of the locus, especially in Chinese populations. The AMELX / AMELY system has also been modified to perform sex determination in non-human primates. It has been successfully applied to primates closely related to humans but does not work on distant relatives such as orangutans or baboons. Due to the inability to generalize the use of amelogenin in non-human primates, several alternative methods have been developed. For most of them X and Y related fragments were included in addition to the AMELX / AMELY test. However, most of these typing methods have been based on human references and despite positive amplification in great apes because of their close relatives, they do not work in more distant primate species.

In addition to the AMELX / AMELY trials, a recent study identified the UTX / UTY locus as a better candidate system for a universal primate sexing marker by comparing genomic data from several mammals. This sex test gives positive results on baboons and orangutans. However, it presents a risk of error due to the large structural variations existing on the Y chromosome and the associated probability of zero amplification of the Y allele. When a Y target locus can not be amplified, this may lead to poor identification of sex.

 The present invention also relates to a method for determining the sex of a DNA sample which comprises a step of amplifying the SRY, UTY and UTX genes of the DNA sample.

Amplification of UTX only in the absence of SRY and UTY indicates that the DNA sample is from a female subject.

 The amplification of the 3 markers, UTY, SRY and UTX indicates that the DNA sample is from a male subject.

 The amplification of 2 markers, UTY, and UTX, in the absence of SRY, indicates that the DNA sample is from a male subject, but that there is a mutation at the hybridization site of SRY primers, or a deletion of this gene, not allowing its amplification (frequent case which is often specific population).

The amplification of 2 markers, SRY, and UTX, in the absence of UTY, indicates that the DNA sample is from a male subject, but that there is a mutation at the hybridization site of UTY primers, or a deletion of this gene, not allowing its amplification (frequent case which is often specific population). The method of typing sex according to the invention is applicable to prevent misidentification of sex. In addition, it is easily applicable for a broad spectrum of DNA quality.

 This method for determining the sex of a subject makes it possible, in addition to identifying the sex, to indirectly evaluate the degradation profile of the male DNA sample studied.

 Indeed, the 3 markers having respective sizes of 81 (SRY), 91 (UTY) and 120 (UTX) bp for the human and chimpanzee sexing tool and 91 (UTY), 120 (UTX) and 165 (SRY) ) for the all primate sexing tool, the decreasing amplification (if any) of these 3 markers tells us about the degree of fragmentation of the sample. This is particularly interesting when using this method of sex determination before determining the genetic profile of a subject further.

 Thus, by proceeding in advance with this step, it is possible to quickly evaluate whether the sample is suitable for being analyzed or not.

 In a preferred embodiment, the step of amplifying the SRY, UTY, and UTX genes is the amplification of 80 to 250 base pair fragments of SRY, UTX, and UTY exon.

 Since exons are coding parts, they are more likely to remain conserved over time in the same species but also between species.

 The amplified fragment may especially be between 80 and 250 base pairs, 80 and 200 base pairs or 80 and 150 base pairs.

 The present invention also relates to a kit for determining the sex of a DNA sample in which the sex determination means comprises SRY, UTY and UTX gene amplification means. Sexing tool of 'All Primates':

 According to one embodiment of this method, the DNA sample is a DNA sample belonging to the order of primates.

 The DNA sample may for example come from a human (Homo sapiens), Pan sp., Gorilla sp., Pongo sp. Hylobates sp., Cercopithecus sp., Erythrocebus patas, Cercocebus torquatus, Mandrillus sphinx, Lophocebus albigena, Papio sp., Macaca sp., Alouatta caraya, Saimiri sciureus, Cebus apella, Aotus trivirgatus, Callithrix jacchus, Hapalemur sp., Eulemur sp. , Microcebus sp., Lepilemur sp., Avahi sp., Propithecus sp., Lemur sp., Indri indri.

In particular, the DNA sample may come from Homo sapiens, Pan troglodyte troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus pygmaeus, Lar larob, Cercopithecus cephus, Cercopithecus nictitans, Erythrocebus patas, Cercocebus torquatus, Mandrillus sphinx, Lophocebus albigena , Macaca mulatta, Macaca sylvanus, Alouatta caraya, Aotus trivirgatus, Sirmiri sciureus, Cebus apella, Callithrix jacchus, Lepilemur dorsalis, Lepilemur sahamalazensis, Lepilemur ankaranensis, Lepilemur mittermeieri, Lepilemur ruficaudatus, Lepilemur aeeclis, Microcebus murinus, Hapalemur griseus ranomafanensis, Hapalemur griseus meridionalis , Hapalemur griseus occidentalis, Hapalemur griseus griseus, Hapalemur aureus, Hapalemur simus, Eulemur macaco macaco, Eulemur macaco flavifrons, Lemur catta, Propithecus diademma edwardsi, Propithecus tattersalli, Propithecus verreauxi coquerelli, Propithecus verreauxi verreauxi, Propithecus verreauxi deckeni, Propithecus diademma diademma, Avahi laniger, Avahi occidentalis, Avahi cleesei, Indri indri.

 In one embodiment, the step of amplifying the SRY, UTY, and UTX genes is the amplification of 100 to 250 base pair fragments of an exon of SRY, UTX, and

UTY.

 In one embodiment, the step of amplifying the genes comprises adding primer pairs having the nucleic sequences:

 SEQ ID NO: 70 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

 and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

 The sequences of the primers of the various preferred embodiments of the sex determination method are given in Table 4 below.

 Table 4

In one embodiment, the kit is a kit for determining the sex of a primate DNA sample and the SRY, UTY, and UTX gene amplification means comprises the primer pairs having the nucleic sequences:

 SEQ ID NO: 70 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

 and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

Typically, the primers hybridize under stringent conditions. For example, for primers of 15 to 25 bp, stringent conditions are a temperature of 55 ° C in buffer or 51 ° C in this same buffer for prosimian samples. The buffer may for example be composed of Multiplex PCR Master mix 2X comprising optimized concentrations (it is they who say it!) Of HotStarTaq® Plus DNA Polymerase, MgCI2, and dNTPs Mix (dATP, dCTP, dGTP, dTTP ultrapure quality ) and Multiplex PCR Plus Buffer (contains 6 mM MgCl2; pH 8.7), Q-solution 5X, CoralLoad Dye 10X, RNase-free water ultra-pure quality, PCR-grade as in the kit QIAGEN Multiplex PCR Plus Kit (ref: 206152).

Tool for human sex and chimpanzees:

According to one embodiment of this method, the DNA sample is a sample of human DNA or chimpanzee.

 According to one embodiment of this method for determining the sex of a DNA sample, the DNA sample is from a human or a chimpanzee and the gene amplification step includes adding primer pairs selected from the group consisting of:

 - for SRY:

 a primer meaning between 15 and 25 base pairs hybridizing between the bases 2,655,350 and 2,655,400, preferably between the bases 2,655,365 and 2,655,395, more preferably between the bases 2,655,369 and 2; 655,390 of the negative strand of the human Y chromosome and

 an antisense primer between 15 and 25 base pairs hybridizing between the bases 2655

295 and 2,655,340 preferably between bases 2655,300 and 2,655,325, more preferably between bases 2655,304 and 2,655,323 of the positive strand of the human Y chromosome,

 a primer meaning between 15 and 25 base pairs, hybridizing between the bases 26 202 990 and 26 203 035, preferably between the bases 26 202 995 and 26 203 030, more preferably between the bases 26 203 002 and 26203023de the strand positive of the Chimpanzee Y chromosome and

 an antisense primer between 15 and 25 base pairs hybridizing between the bases 206 203 055 and 26 203 105, preferably between the bases 26 203 060 and 26 203 095, more preferably between the bases 26 203 069 and 26,203,088 of the negative strand of the chimpanzee Y chromosome,

-for UTX:

 a primer meaning between 15 and 25 base pairs hybridizing between the bases 44 929 400 and 44 929 450, preferably between the bases 44 929 415 and 44 929 440, more preferably between the bases 44 929 417 and 44 929,437 of the positive strand of human chromosome X and

 an antisense primer between 15 and 25 base pairs hybridizing between the bases 44 929 505 and 44 929 555, preferably between the bases 44 929 515 and 44 929 550, more preferably between the bases 44 929 521 and 44,929,541 from the negative strand of the human X chromosome,

a primer meaning between 15 and 25 base pairs hybridizing between the bases 45 534 530 and 45 534 580, preferably between the bases 45 534 535 and 45 534 570, more preferably between the bases 45 534 544 and 45 534,564 from the positive strand of chimpanzee X chromosome and an antisense primer between 15 and 25 base pairs hybridizing between the bases 45 534 630 and 45 534 800, preferably between the bases 45 534 535 and 45 534 575, more preferably between the bases 45 534 648 and 45,534,668 of the negative strand of chimpanzee X chromosome,

and

 -for UTY:

 a primer meaning between 15 and 25 base pairs hybridizing between the bases 447 590 and 447 645, preferably between the bases 447 600 and 15 447 635, more preferably between the bases 447 608 and 15 447 605; 447,628 of the negative strand of the human Y chromosome and

 an antisense primer between 15 and 25 base pairs that hybridizes between the bases 447 520 and 44 44 560, preferably between the bases 447 530 and 15 44 7 550, more preferably between the bases 447,533 and 15,447,553 of the positive strand of the human Y chromosome

 a primer of between 15 and 25 base pairs hybridizing between bases 860 260 and 20 860 310, preferably between bases 860 270 and 20 860 300, more preferably between bases 860 275 and 2090. 860,295 of the negative strand of chimpanzee Y chromosome and

 an antisense primer between 15 and 25 base pairs hybridizing between the bases 860 185 and 20 860 235, preferably between the bases 860 195 and 20 860 225, more preferably between the bases 860 200 and 20,860,220 of the positive strand of the chimpanzee Y chromosome.

Typically, the primers hybridize under stringent conditions. For example, for primers of 15 to 25 bp, stringent conditions are a temperature of 55 ° C in buffer or 51 ° C in this same buffer for prosimian samples. The buffer may for example be composed of Multiplex PCR Master mix 2X comprising optimized concentrations of HotStarTaq® Plus DNA Polymerase, MgCl2, and dNTPs Mix (dATP, dCTP, dGTP, dTTP ultrapure quality) and Multiplex PCR Plus Buffer (contains 6 mM MgCl2 pH 8.7), Q-solution 5X, CoralLoad Dye 10X, RNase-free water ultra-pure quality, PCR-grade as in the kit QIAGEN Multiplex PCR Plus Kit (ref: 206152).

In one embodiment, the step of amplifying the genes comprises adding primer pairs having the nucleic sequences:

 SEQ ID NO: 70 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

 and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

or

 SEQ ID NO: 65 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY

and SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

 In a preferred embodiment, the step of amplifying the genes comprises adding primer pairs having the nucleic sequences:

 SEQ ID NO: 65 and SEQ ID NO: 66 for SRY,

 SEQ ID NO: 67 and SEQ ID NO: 68 for UTY and

 SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.

The sequences of the primers of this preferred embodiment are given in Table 5 below.

 Table 5

This last embodiment of the sex typing method is only applicable to a limited number of primates but it allows to use amplicons of very small sizes (between 80 and 120 bp) and therefore to work on the DNA is highly degraded.

The invention will be illustrated by the following figures and examples. However, by way of illustration, these examples and figures should not be interpreted as limiting the scope of the present invention. FIGURES

 Figure 1 shows a comparison between the mutability and the discriminating power of existing multiplexes and those of the invention (CombYpIex).

 Figure 2 shows an example of a human profile obtained from multiplex M1.

 For each of the profiles, the horizontal axis represents the size of the DNA fragments: on the left the small sizes, on the right the largest sizes. Each peak corresponds to a STR marker.

Each line corresponds to a fluorochrome. The gray bar above each peak is named after the marker below, and its length reflects the spectrum of all expected alleles (polymorphism). The place of each potentially amplified allele is represented as a gray bar.

 Figure 3 shows an example of a human profile obtained from the multiplex M2 combined with a sexing tool. The reading codes are the same as before.

Figure 4 shows an example of a chimpanzee profile obtained from the multiplex M1. The reading codes are the same as before. Figure 5 shows an example of a chimpanzee profile obtained from multiplex 2 combined with a sexing tool. The read codes are the same as before. FIG. 6 represents an example of a male profile with a UTY peak, a UTX peak and an SRY peak (profile A).

Figure 7 shows an example of a male profile with a UTY peak, a UTX peak but no SRY peak (B profile).

 Figure 8 shows an example of a male profile with a UTX peak and an SRY peak but no SRY peak (C profile).

 Figure 9 shows an example of a female profile with only a UTX peak (profile D). Figure 10 shows the results of the human / chimpanzee comparison tests as a dendogram.

 Figure 11 shows the PCA of the comparative men / chimpanzee tests.

 Figure 12 shows a dendrogram of the results obtained in the analysis of Feakes and its derivatives, Attenborough, Starbuck and Revis.

EXAMPLES

EXAMPLE 1: Determination of the genetic profile of a subject

 Development of reference sequences

A comprehensive list of STR markers on the human Y chromosome has been established based on available literature (Goedbloed et al 2009, Hanson et al 2007, Ballantyne et al 2010, Vermeulen et al 2009, Maybruck et al. 2009, Hanson et al., 2012, Kayser et al., 2004). and completed a work with use of databanks ncbi, ucsc, together.

From this list, a study was conducted to gather all the information relating to each listed marker, which could provide information on their discriminant potential (molecular structure, mutation rate, localization in the genome, evidence of duplication , etc.).

 This same information was collected for the chimpanzee and the markers absent in this species, or which were not suitable for the chimpanzee, were eliminated.

A ranking of markers according to their mutation rates, molecular structure, quality of flanking regions, and others, has been achieved. The selected markers were divided into 2 multiplexes: the M1, comprising the markers having a mean mutation rate (see Table 6A and 6B), and the 2, comprising the markers having a high mutation rate (see Table 7A and 7B).

Table 6A

Table 6B

Table 7A

Table 7B

Design of multiplex primers.

 The theoretical diagram of the 2 multiplexes was realized, by organizing the markers per line (1 line = 1 fluorochrome) according to their sizes in order to ensure a good distribution of the markers on the range of fixed size (from 80 to 350 bp) .

 Alignment of about 500 bp sequences around the human-chimpanzee marker was performed for each marker. A pair of primers capable of amplifying the STR marker for both species, and for a given (near) size, was selected. The primers were modified until the designated theoretical pattern was reached / obtained, and in the case where this was not possible, the theoretical scheme was modified until satisfied.

 For each marker, an amplification was carried out using the cold primers for a male and a female individual, for the man and the chimpanzee, to see if a band of the expected size was obtained, and especially if this one was unique in order to avoid a-specificities and duplications that would not have been reported or detected until then. The primers were remodified until they reached satisfaction.

 Once all the markers were independently tested, they were multiplexed per line (ie fluorochrome) to detect potential interactions.

 The markers were then labeled labeled ('hot'), and again amplification was performed using warm primers for a male and a female individual, for both humans and chimpanzees, to see if we were getting a band at the expected size, and especially if it was unique to avoid a-specificities and duplications. The primers were remodified until they reached satisfaction.

 Final multiplexing was performed, including all markers and the concentrations were adjusted to a balanced multiplex.

The same operation was repeated for the second multiplex.

 In parallel, each marker was sequenced in order to establish a correspondence between size of the amplicon by genotyping and the actual number of repetitions corresponding to this size.

Table 8A below summarizes the information on the markers 18 ¹ multiplex M1 for human samples.

Table 8B below summarizes the information on the markers 18 ¹ multiplex M1 for samples of chimpanzees.

Table 9A below summarizes information on 14 markers of 2 ^nd multiplex 2 and for sex markers (SRY, UTY and UTX) for human samples.

Table 9B below summarizes information on the 14 markers of the 2 ^nd multiplex M2 and for the sex markers (SRY, UTY and UTX) for chimpanzee samples.

Table 8A: Information on the 18 markers of the Multiplex M1 for human samples.

Table 8B: Information on the 18 markers of the M1 Multiplex for chimpanzee samples.

Table 9A: Information on the 14 markers of the M2 multiplex and for SRY, UTY and UTX for human samples.

Table 9B: Information on the 14 markers of the M2 multiplex for chimpanzee samples (NA for not available)

Test of M1 and M2 multiplexes on men and chimpanzees: choice of samples to maximize the observed genetic variability.

 A sample panel of 13 men and 11 chimpanzees were tested. They were genotyped with the 2 multiplexes in order to obtain an idea of the genetic variability detected (i.e. alleles) for the M1 and M2 multiplexes, for the men and the chimpanzees.

 Men come from different countries and are therefore likely to have very different genetic profiles - this should allow for maximum genetic variability and an idea of the expected range of alleles. Chimpanzees come from various zoos and collections, to avoid the effects of consanguinity, and in the same way, to maximize our chances of getting different profiles.

Results:

The degree of polymorphism (variability) of the markers included in the 2 multiplexes was estimated for the panel. The results are given in Table 10 for M1, in Table 11 for M2 and in Table 12 for the sexing tool. It is observed that the number of alleles and the variance is in agreement with the mutation rates predicted for M1 and M2, ie a larger variance for M2 (high mutation rate) than for M1.

Table 10

M2 Y GATA A10 DYS570 DYS549 DYS460 DYS442

Samples Allele Size Allele Size Allele Size Allele Size Allele Size

Hum_FX 13 163.55 18 229.95 12 299.49 12 1 3.52 12 181, 39

Hum_20FT 12 159.45 15 217.57 12 299.49 10 105.19 1 1 177.4

Hum_21 PF 14 167.59 20 238.12 12 299.56 11 109.41 13 185.44

Hum_28IT 12 159.45 18 229.93 12 299.56 10 105.18 13 185.48

Hum_29AH 13 163.56 18 229.88 13 303.43 10 105.23 1 1 177.4

Hum_310L 13 163.6 17 225.85 13 303.47 10 105.28 12 181, 45

Hum_34AHB 13 163.53 16 221, 7 12 299.49 11 109.39 13 185.44

Hum_35JN 12 159.46 18 229.83 13 303.47 10 105.29 1 1 177.27

Hum_38ME 12 159.46 16 221, 77 12 299.42 11 109.42 1 1 177.36

Hum_39TB 13 163.52 18 229.84 12 299.63 11 109.29 12 181, 41

Hum_40EH 12 159.44 17 225.8 12 299.63 11 109.33 12 181, 42

Hum_43PF 12 159.36 19 233.99 12 299.7 11 109.36 12 181, 49

Hum_45AMB 12 159.44 22 246.47 1 1 295.64 11 109.34 12 181, 42

Hum César 13 163.63 18 229.95 13 303.46 10 105.17 12 181, 43

Chimp 103 4 130.85 4 176.18 8 283.6 10 105.08 1 1 189.51

Chimp_211 4 130.8 4 176.06 10 291, 54 10 105.05 1 1 189.48

Chimp 253 4 130.79 4 176.11 8 283.53 9 100.82 1 1 189.53

Chimp_273 4 130.75 4 176.16 8,283.55 11,109.15 1 1,189.47

Chimp_295 4 130.83 4 176.17 8 283.56 10 105.01 1 1 189.54

Chimp_367 4 130.84 4 176.19 9 287.58 10 104.98 1 1 189.47

Chimp_61A 4 130.84 4 176.11 8 283.61 11 109.18 10 185.5

Chimp_Sg229 4 130.73 4 176.18 8 283.62 10 105.03 1 1 189.51

Chimp_Sg369 4 130.82 4 176.19 10 291, 66 10 104.99 1 1 189.55

Chimp SgCI 4 130.85 4 176.19 8 283.59 11 109.13 12 193.61

Chimp_202

 0 0 0 0 0 0 0 0 0 0 (female / control)

 Nb alleles Ho 3 7 3 3 3

 Allelic Range 12-14 15-22 11-13 10-12 11-13

Nb alleles Chz 1 1 2 3 3

Allelic Range (4) (4) 8-10 9-11 10-12

Table 1 1

2 SRY UTX UTY

 Sam Pies Allele Size Allele Size Allele Size

 Hum_FX 1 81.56 1 120.61 1 91.36

 Hum_20FT 1 81.57 1 120.61 1 91.4

 Hum_21PF 1 81.67 1 120.62 1 91.44

 Hum_28IT 1 81.57 1 120.61 1 91.4

 Hum_29AH 1 81.56 1 120.62 1 91.36

 Hum_310L 1 81.63 1 120.68 1 91.43

 Hum_34AHB 1 81.57 1 120.61 1 91.45

 Hum_35JN 1 81.56 1 120.61 1 91.4

 Hum_38ME 1 81.54 1 120.6 1 91.38

 Hum_39TB 1 81.58 1 120.62 1 91.42

 Hum_40EH 1 81.67 1 120.62 1 91.38

 Hum_43PF 1 81.6 1 120.62 1 91.43

 Hum_45AMB 1 81.6 1 120.63 1 91.4

 Hum_Cesar 1 81.64 1 120.61 1 91.45

 Chimp_103 1 81.59 1 120.7 1 91.34

 Chimp_211 1 81.57 1 120.62 1 91.28

 Chimp_253 1 81.56 1 120.61 1 91.36

 Chimp_273 1 81.68 1 120.63 1 91.35

 Chimp_295 1 81.6 1 120.71 1 91.31

 Chimp_367 1 81.59 1 120.7 1 91.3

 Chimp_61A 1 81.59 1 120.7 1 91.34

 Chimp_Sg229 1 81.59 1 120.63 1 91.34

 Chimp_Sg369 1 81.59 1 120.62 1 91.3

 Chimp SgCI 1 81.67 1 120.62 1 91.39

 Chimp_202_Fem 0 1 120,7 0

 Nb alleles Ho 1 1 1

 Nb alleles Chz 1 1 1

 Table 12

 These tables summarize all the genetic variations (also called alleles) observed in individuals, and for each marker. We quickly see that each marker has several alleles in humans and a little less in chimpanzees - a result perfectly compatible with the demographic history of these 2 species. These results, which relate only to 13 and 11 individuals, are already proving more effective than what has been published so far in chimpanzees. It is therefore very revealing of the power of discrimination of this tool.

 These results shown as a dendrogram in Figure 10 and analyzed by PCA in Figure 11 show that Combyplex allows unambiguous discrimination of both species.

The CombYpIex assembly which includes the M1 and M2 multiplexes shows a very good reproducibility and robustness, both in the man and in the chimpanzee. It can amplify 30/31 markers with sizes always less than 350 bp, it allows to make moderately degraded DNA, as tested on individuals dating from the 16th century. Discriminant capacity of CombYplex compared to AmplYfiler: Individuals same last names:

 CombYplex has been used on individuals with the same family name or derivatives of this name (Feakes and derivatives, Attenborough, Starbuck and Revis) to test whether this tool discriminates individuals having a priori recent common origin (establishment last names to 15sc). The results of this study are presented as a dendogram in Figure 12.

These results show that CombYplex fingerprints unambiguously allow individuals with the same surnames to be discriminated: individuals are distinctly divided into 4 groups (4 branches of the dendrogram) corresponding to the 4 surnames studied. This indicates a direct link between Y chromosome and surname; In this case, it also indicates that the patronymic variants of the 4 surnames chosen, are recent and of multiple origins since they are distributed randomly within the patronymic groups, but do not form distinct subgroups that could have suggested common origin. Two individuals are excluded from the tree clearly indicating an incompatibility between the name and the Y chromosome in question, since they do not fit into any of the four probable branches - this is the case between a paternity name or an adoption case; the rate varies from 2 to 4% in England, this observation is perfectly compatible with statistics on the subject.

The CombYplex tool makes it possible to group individuals with a common surname and having a common origin, but also to detect a non-congruence between family name and Y chromosome and is a valuable ally in genealogical / patronymic analyzes.

Descriptive statistics :

The following formulas have been used: (1) discriminant capacity = no. of individuals / no. the different haplotypes observed; (2) gene diversity (h), equivalent to the expected frequency of heterozygous frequencies with autosomal diploid loci was calculated as (η / (η-1)) · (1-Σρ, ² ), where pi = frequency from the allele to the ith.

EXAMPLE 2: Sexa e tool

Materials and methods

 Samples of human and non-human primates.

 A panel of 177 DNA samples was used, including 1 16 human samples, and 61 non-human primate samples from 45 different species. Human samples were obtained from healthy volunteers and samples of non-human primates came from a collection.

A list of these samples, including the taxonomic group, the common name of the species, and my previously obtained sex information is given in Table 13. DNA samples used were extracted from different substrates including saliva, serum, whole blood, tissue and cell culture.

Table 13

DNA extraction

 The DNA extraction process depends on the type of sample.

The DNA was extracted from:

- (i) whole blood using the QiaAmp DNA Blood mini kit (Qiagen),

 - (ii) serum using the mini-kit i-genomic DNA Blood mini-kit (Euromedex)

 (iii) saliva using the OG-300 Oragene DNA Self-Collection Kit (DNA Genotek) kit,

- (iv) cell culture using the Blood & Cell Culture DNA mini kit (Qiagen), according to the manufacturer's instructions for each of the kits used.

The quantity and quality of the extracted DNA was estimated using a NanoDrop spectrophotometer.

PCR primers design

 The PCR primers for the amplification of the SRY fragment have been published previously (Fiore et al., 2005). PCR primers for co-amplification of UTX and UTY homologous regions were designed from conserved UTX / UTY regions. Sequences available in libraries for different primate species were aligned using BioEdit v.7.0.5.3. The conserved regions for each gene were used for primer design using Primer3 software v.0.4.0 to allow co-amplification of relatively small UTX and UTY fragments from sample probably comprising degraded DNA. 5'-labeled sense primers with a fluorescent FAM fluorochrome (InVitrogen) were used to allow detection by capillary electrophoresis. The sequences of the primers and the sizes of the amplicons are shown in Table 6.

Primer meaning Anti-sense primer Length

Enhanced SEQ ID SEQ ID marker

 Sequence (5'-3 ') Sequence (5'-3')

 NO NO (bp)

 tgtgcctcctggaagaatgg

 SRY 70 agtgaagcgacccatgaacg

 66,165

UTY cagtgttaccag ccttaacag ggcaggtctacttttgtagag 91

 67 68

 UTX

 tctgtggactaggtttgtggt 120

69

Table 6

Amplification by multiplex PCR

Regions of UTX, UTY and / or SRY were simultaneously amplified in a final volume of 12.5 μl consisting of 1X of PCR buffer B (Fisher Bioblock), 1.5 mM of MgCl ₂ , 200 μl of each dNTP, 0. , 2 to 0.4 μl of primers, 1 U of Taq DNA polymerase (Fisher Bioblock) and 15 to 25 ng of DNA sample. The concentrations of the primers for each primer are given in Table 6. The PCR cycle conditions are a first denaturation step at 94 ° C for 5 min followed by 40 denaturation cycles at 94 ° C for 30 s, hybridization to 55 ° C for 30s and an elongation at 72 ° C for 30s with a final elongation step at 72 ° C for 5min. It is important to note the hybridization temperature was decreased to 51 ° C for prosimian samples.

Electrophoresis detection

 The PCR products were first visually assayed by agarose gel electrophoresis (140V, 40min) on 2% agarose gels using SYBR Safe staining (Invitrogen). For the preparation of the samples for capillary electrophoresis detection, 1 μl of 100-fold diluted PCR products were then mixed with 0.2 μΙ internal standard GeneScan-600LIZ and 8.8 μΙ formamide Hi-Di. After denaturation at 95 ° C for 5 min, the samples were separated on an ABI 3730 Genetic Analyzer using POP-7. The data was analyzed using GeneMapper v. 4.0.

Sequencing

 UTX, UTY and SRY were separately amplified using the primer pairs in Table 6, as previously described. Each PCR product was sequenced in two reactions using the sense and antisense PCR primers. The sequencing reaction was performed with a Big Dye Terminator v3.1 sequencing kit (Applied Biosystems). The PCR products were separated on an ABI 3730 Genetic Analyzer (Applied Biosysems). The sequences were analyzed and assembled using Sequence Scanner v1.0 software (Applied Biosystems) and the BioEdit Sequence Alignment Editor.

Results

 Multiplex PCRs developed including both UTY UTY and SRY primers allow for sex determination for each DNA sample tested. The results are given in Table 7.

Female Male

Profile F ^" Profile A Profile B Profile C

Hominidae Homo sapiens 2 2 0 0 Pan sp. 2 3 0 0

Gorilla sp. 1 2 0 0

Pongo sp. 1 2 0 0

Hylobatidae Hylobates sp. 2 2 0 0

Cercopithecidae Cercopithecus 1 1 0 0 sp.

 Erythrocebus 1 0 0 0 patas

 Cercocebus 0 1 0 0 torquatus

 Mandrillus 0 1 0 0 sphinx

 Lophocebus 1 0 0 0 albigena

 Papio sp. 1 1 0 0

Macaca sp. 1 2 0 0

Atelidae Alouatta 0 1 0 0 caraya

 Cebidae Saïmiri 0 0 0 3 sciureus

 Cebus apella 1 0 0 1

Aotus 0 1 0 0 trivirgatus

 Callithrix 0 1 0 0 jacchus

 Lemuridae Hapalemur 6 7 0 0 sp.

 Eulemur sp. 0 3 0 1

Lepilemuridae Lepilemur sp. 2 4 2 0

Indriidae Avahi sp. 3 1 1 0

 Propithecus 4 2 0 0 sp.

 Indri indri 0 0 0 1

Table 7

Female samples were identified by a single signal corresponding to 125 bp specific UTX fragments (F profile, Figure 9). On the contrary, three different profiles were observed for male samples as illustrated in Figures 6 to 8. In fact, the male samples were identified either by three signals, which corresponded to the specific fragment of X and the two specific fragments of Y (Profile A) or occasionally by two signals which corresponded to the specific fragment of X and only to one of the two fragments of the Y chromosome that is to say either SRY (C profile) or UTY (B profile). The Results obtained for each sample are given in Table 5. A typical male profile (Profile A) with both SRY and UTY fragments was observed for most samples. The six samples that did not have UTY fragments but clearly showed a specific SRY signal belong to the New World monkey species (Saïmri sciureus (n = 3) and Cebus apella (n = 1)) and to both species of prosimians. (Indri Indri (n = 1) and Eulemur macaco flavifrons (n = 1)) The five samples that did not show a SRY fragment but showed a specific signal of clear UTY belonged to the old-world monkey species (Lophocebus albigena). ) and four species of prosimians.

 For the 54 samples tested whose sex was already known, the sex determined with the new test was always consistent with the morphological sex. In order to investigate whether a failure of the amplification of the UTY or SRY locus was due to a mutation in the primer binding site or to a deletion of new UTRY and SRY primer sets were designed. Amplifications with the second sets of primers were successful for all samples and the resulting amplicon sequencing revealed that the inability to amplify the locus with the first set of primers was due to mutations in the binding of the primers.

Thus, the newly developed method unambiguously allows the gender of all tested samples to be determined even in the presence of a mutation in the primer region. The first and main advantage of this method is that it combines two UTX / UTY and SRY independent sexing tests in an assay for exact sex determination. Indeed, 11 male specimens that did not have UTY or SRY fragments that would previously have been considered female samples based on the UTX TUY or SRY locus alone were identified as males by this approach. . This shows the need for a sex determination test that includes at least 2 Y markers for primates, thereby preventing misidentification of sex due to high structural polymorphism on the Y chromosome or mutation at the binding site. the primer.

By using two Y-specific fragments, the negative effect of a mutation on the primer binding site or a deletion on an amplification can be minimized since a mutation and / or deletion on both loci is unlikely to occur. The second advantage of this method is its wide spectrum of application in primates. Indeed, it works on all species tested especially through the work on the design of primers. The same method works on all the species tested, but a slight optimization by reducing the hybridization temperature is preferable for sexing the distant prosimic species. This method is accessible to molecular biology laboratories with basic equipment since the size difference between the three possible amplification products is large enough to be discerned by simple agarose gel electrophoresis and an automatic sequencer is not necessary. Finally, the small size of the amplification products (<200bp) makes them useful for sex identification of potentially degraded samples such as serum samples and also non-invasive samples such as hair or faeces.

REFERENCES

During this application, different references describe the state of the art of the invention. The descriptions of these references are hereby incorporated by reference in the present description.

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Claims

1. Method for characterizing a human or chimpanzee DNA sample comprising a step of: -determination of the alleles of at least 10 STR loci of the DNA sample, the loci being chosen from group M2, in which : -group M2 consists of: Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533. 2. Method for characterizing a DNA sample according to claim 1 further comprising the step of determining the alleles of at least 10 STR loci chosen from the M1 group, in which : -group M1 consists of: DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538. 3. Method for characterizing a DNA sample according to claim 1 or 2 comprising determining alleles from 10 to 50 STR loci, preferably alleles from 10 to 40 STR loci, more preferably alleles from 10 to 32 STR loci. 4. Method for characterizing a DNA sample according to any one of claims 1 to 3 comprising the step of determining the alleles of 14 STR loci chosen from group M2. 5. Method for characterizing a DNA sample according to any one of claims 1 to 4 comprising the determination of the alleles: -18 STR loci chosen from group M1 And -14 STR loci chosen from group M2. 6. Method for characterizing a DNA sample according to any one of claims 1 to 5 comprising a sex determination step in which the sex determination comprises the step of amplifying the SRY, UTY and UTX genes of the DNA sample. 7. Method for characterizing a DNA sample according to claim 6 in which the step of amplifying the SRY, UTY and UTX genes comprises the addition of pairs of primers having the nucleic sequences: SEQ ID NO: 65 and SEQ ID NO: 66 for SRY, SEQ ID NO: 67 and SEQ ID NO: 68 for UTY And SEQ ID NO: 67 and SEQ ID NO: 69 for UTX. 8. Kit comprising means for determining the alleles of at least 10 STR loci of a human or chimpanzee DNA sample, the loci being chosen from group M2, in which: -group M2 consists of: Y-GATA-A10, DYS570, DYS549, DYS460, DYS442, DYS510, DYS541, DYS576, DYS513, DYS458, DYS481, DYS612, DYS444 and DYS533. 9. Kit according to claim 8 comprising at least 10 pairs of primers suitable for amplifying the STR loci and in which the primer pairs have nucleic sequences chosen from the group consisting of: SEQ ID NO: 37 and SEQ ID NO: 38 for the Y-GATA-A10 locus SEQ ID NO: 39 and SEQ ID NO: 40 for the DYS570 locus, SEQ ID NO: 41 and SEQ ID NO: 42 for the DYS549 locus, SEQ ID NO: 43 and SEQ ID NO: 44 for the DYS460 locus, SEQ ID NO: 45 and SEQ ID NO: 46 for the DYS442 locus SEQ ID NO: 47 and SEQ ID NO: 48 for the DYS510 locus, SEQ ID NO: 49 and SEQ ID NO: 50 for the DYS541 locus, SEQ ID NO: 51 and SEQ ID NO: 52 for the DYS576 locus, SEQ ID NO: 53 and SEQ ID NO: 54 for the DYS513 locus, SEQ ID NO: 55 and SEQ ID NO: 56 for the DYS458 locus, SEQ ID NO: 57 and SEQ ID NO: 58 for the DYS481 locus SEQ ID NO: 59 and SEQ ID NO: 60 for the DYS612 locus, SEQ ID NO: 61 and SEQ ID NO: 62 for the DYS444 locus and SEQ ID NO: 63 and SEQ ID NO: 64 for the DYS533 locus. 10. Kit according to claim 8 or 9 comprising, in addition, means for determining the alleles of at least 10 STR loci chosen from the M1 group, in which : -group M1 consists of: DYS485, DYS588, DYS502, DYS461, DYS638, DYS643, DYS587, DYS575, DYS578, DYS632, DYS508, DYS640, DYS511, DYS577, DYS556, DYS517, DYS565 and DYS538. 1 1. Kit according to claim 10 comprising at least 10 pairs of primers suitable for amplifying the STR loci and in which the primer pairs have nucleic sequences chosen from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2 for the DYS485 locus, SEQ ID NO: 3 and SEQ ID NO: 4 for the DYS588 locus, SEQ ID NO: 5 and SEQ ID NO: 6 for the DYS502 locus, SEQ ID NO: 7 and SEQ ID NO: 8 for the DYS461 locus, SEQ ID NO: 9 and SEQ ID NO: 10 for the DYS638 locus, SEQ ID NO: 1 1 and SEQ ID NO: 12 for the DYS643 locus, SEQ ID NO: 13 and SEQ ID NO: 14 for the DYS587 locus, SEQ ID NO: 15 and SEQ ID NO: 16 for the DYS575 locus, SEQ ID NO: 17 and SEQ ID NO: 18 for the DYS578 locus, SEQ ID NO: 19 and SEQ ID NO: 20 for the DYS632 locus, SEQ ID NO: 21 and SEQ ID NO: 22 for the DYS508 locus, SEQ ID NO: 23 and SEQ ID NO: 24 for the DYS640 locus, SEQ ID NO: 25 and SEQ ID NO: 26 for the DYS511 locus, SEQ ID NO: 27 and SEQ ID NO: 28 for the DYS577 locus, SEQ ID NO: 29 and SEQ ID NO: 30 for the DYS556 locus, SEQ ID NO: 31 and SEQ ID NO: 32 for the DYS517 locus, SEQ ID NO: 33 and SEQ ID NO: 34 for the DYS565 locus, SEQ ID NO: 35 and SEQ ID NO: 36 for the DYS538 locus. 12. Kit according to any one of claims 8 to 11 comprising means for determining the alleles of 10 to 50 STR loci, preferably the alleles of 10 to 40 STR loci, more preferably the alleles of 10 to 32 STR loci. 13 Kit according to any one of claims 8 to 12 comprising means for determining the alleles of at least 14 STR loci chosen from group M2. 14. Kit according to any one of claims 8 to 13 comprising means for determining the alleles: -18 STR loci chosen from group M1 And -14 STR loci chosen from group M2. 15. Kit according to any one of claims 8 to 14 comprising means for determining sex, in which the means for determining sex consist of means for amplifying the SRY, UTY and UTX genes. 16. Kit according to claim 15 in which the means for amplifying the SRY, UTY and UTX genes comprise pairs of primers having the nucleic sequences: SEQ ID NO: 65 and SEQ ID NO: 66 for SRY, SEQ ID NO: 67 and SEQ ID NO: 68 for UTY And SEQ ID NO: 67 and SEQ ID NO: 69 for UTX.