WO2021018802A1 - Procédé de profilage génétique - Google Patents

Procédé de profilage génétique Download PDF

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WO2021018802A1
WO2021018802A1 PCT/EP2020/071050 EP2020071050W WO2021018802A1 WO 2021018802 A1 WO2021018802 A1 WO 2021018802A1 EP 2020071050 W EP2020071050 W EP 2020071050W WO 2021018802 A1 WO2021018802 A1 WO 2021018802A1
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primer
sequence
nucleotide residues
primers
str
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Ark-Biodiversity Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention is in the field of molecular biology and in particular relates to means and methods for genetic profiling, e.g. for forensic uses. Particularly a method for amplifying nuclei acid sequences comprising Short Tandem Repeat Sequences (STR) is provided.
  • STR Short Tandem Repeat Sequences
  • Genetic profiling or fingerprinting is for example currently used for forensic purposes, in the determination of genetic relationships and/or for identifying individual subjects.
  • One application of genetic fingerprinting is the illegal trade with wildlife including trafficking of protected or endangered animals and plants.
  • a major problem in preventing illegal animal trade is that it has so far not been possible to develop a single specific test for each of the relevant authorities (globally, e.g. CITES, EU, etc., or regionally) that are able to address the countless possible criminal or environmental questions.
  • the present invention provides an improved method for the amplification of at least one short tandem repeat (STR) locus in a DNA containing sample.
  • Said method has a higher sensitivity than the methods previously described as is apparent from the enclosed examples and allows for the determination of genetic fingerprints in samples from essentially all animals and plants that have STRs in their genome.
  • the present invention relates to a method for the amplification of at least one short tandem repeat (STR) locus on a double stranded nucleic acid in a sample, the method comprising the steps of
  • amplifying the STR locus in an amplification reaction comprising contacting the sample with a pair for primers, wherein
  • the first primer of said pair of primers comprises a 5’ anchor sequence and a 3’ repeat sequence, wherein
  • the 5’ anchor sequence of said first primer hybridizes under stringent conditions to the nucleic acid sequence immediately downstream of said STR locus on the first strand of said nucleic acid
  • the 3’ repeat sequence of said first primer hybridizes under stringent conditions to the 3’ portion of said STR locus on the first strand of said nucleic acid
  • the second primer of said pair of primers hybridizes under stringent conditions to a sequence downstream of said STR locus on the second strand of said nucleic acid.
  • the invention pertains to a method for the amplification of at least one short tandem repeat (STR) locus on a double stranded nucleic acid in a sample, the method comprising the steps of
  • amplifying the STR locus in an amplification reaction comprising contacting the sample with a pair for primers, wherein
  • the first primer of said pair of primers comprises a 5’ anchor sequence and a 3’ repeat sequence, wherein
  • the 5’ anchor sequence of said first primer is complementary to a nucleic acid sequence immediately downstream of said STR locus on the first strand of said nucleic acid
  • the 3’ repeat sequence of said first primer is complementary to the 3’ portion of said STR locus on the first strand of said nucleic acid
  • the second primer of said pair of primers is complementary to a sequence downstream of said STR locus on the second strand of said nucleic acid.
  • the amplification reaction is a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the first and/or the second primer comprises one or more modified bases, preferably the first and/or second primer comprises one or more locked-nucleic-acid (LNA) nucleotide residues.
  • the first primer may comprise between 2 and 6 LNA nucleotide residues, preferably between 3 and 5 LNA nucleotide residues, most preferably 3 LNA nucleotide residues.
  • the 5’ anchor sequence of the first primer may advantageously comprise between 1 and 4 LNA nucleotide residues, preferably 2 or 3 LNA nucleotide residues.
  • the 3’ repeat sequence of the first primer may advantageously comprise between 1 and 4 LNA nucleotide residues, preferably 1 or 2 LNA nucleotide residues.
  • the methods of the invention can in particularly be used in DNA fingerprinting, e.g. in the context of forensic analysis, paternity testing or in identifying the heritage or the identification of animals. Description of Figures
  • FIGURE 1 Schematic illustration of the amplification method of the present invention.
  • FIGURE 2 Electropherogram of genotype of Individual 1 verified by PP21.
  • the marker D10S1248 is comprised in alleles 14 and 16 (D10S1248 14/16) and the marker SE33 is comprised in alleles 17 and 20 (SE33 17/20).
  • the marker D12S391 is comprised in alleles 18 and 20 (D12S391 18/22).
  • FIGURE 3 Electropherogram of anchor modified D10S1248 assay performed and analyzed using ABI Prism 310 series.
  • DNA sample was taken from Individual 1 (see Fig. 1).
  • the DNA sample was amplified with a OneTaq Hotstart DNA Polymerase (New England Biolabs GmbH).
  • the resulting PCR product was diluted in a 1 : 10 ratio with nuclease free water.
  • X axis of the electropherogram displays the length of the analyzed fragment/amplicon in base pairs and Y axis displays the signal strength in relative fluorescence units (RFU).
  • a peak on the Y axis represents a fluorescence signal strength dependent number of fragments/amplicons at its respective length on the X axis. The respective temperatures can be seen in the left of each panel.
  • FIGURE 4.1 Schematic representation of the primer design targeting the SE33 locus displays the polymorphic STR region (light grey) and the 5’ flanking sequence (white) containing an anchored primer (dark grey).
  • FIGURE 4.2 Electropherogram of anchor modified SE33 assay performed and analyzed using ABI Prism 310 series. DNA sample was taken from Individual 1 (see Fig. 1). The DNA sample was amplified with a OneTaq Hotstart DNA Polymerase with a Gradient PCR program ranging from 60°C to 55°C. The resulting PCR product was diluted in a 1 :4 ratio with nuclease free water.
  • X axis of the electropherogram displays the length of the analyzed fragment/amplicon in base pairs and Y axis displays the signal strength in RFU. A peak on the Y axis represents a fluorescence signal strength dependent number of fragments/amplicons at its respective length on the X axis. The respective temperatures can be seen in the top left of each panel.
  • FIGURE 5 Electropherogram of multiplexed modified D10S1248, D12S391, and SE33 assay performed and analyzed using ABI Prism 310 series.
  • DNA sample was taken from Individual 1 (see Fig. 1) and Individual 2.
  • the DNA sample was amplified with a OneTaq Hotstart DNA Polymerase PCR program.
  • X axis displays the length of the analyzed fragment/amplicon in base pairs and
  • Y axis displays the signal strength in RFU.
  • a peak on the Y axis represents a fluorescence signal strength dependent number of fragments/amplicons at its respective length on the X axis.
  • (A) represents Individual 1 and (B) Individual 2.
  • FIGURE 6 Electropherogram of multiplexed modified D10S1248, D12S391, and SE33 assay performed and analyzed using ABI Prism 310 series).
  • the DNA samples were amplified with a OneTaq Hotstart DNA Polymerase PCR program.
  • the resulting PCR product was diluted in a 1 : 10 ratio with nuclease free water.
  • X axis displays the length of the analyzed fragment/amplicon in base pairs and Y axis displays the signal strength in RFU.
  • a peak on the Y axis represents a fluorescence signal strength dependent number of fragments/amplicons at its respective length on the X axis.
  • The“sz” value represents the length of the fragment, while“ht” is the peak height in RFU.
  • the first generation (A, B) can be seen in the first two panels and they are the parents of (C) who’s genotype is in the third panel. (D)’s parental lineage is not displayed, and the remaining four panels (E, F, G, H) are (C) and (D)’ s children.
  • FIGURE 7 Electropherogram of anchor modified D10S1248 assay performed and analyzed using ABI Prism 310 series.
  • DNA sample was taken from Individual 1 (see Fig. 1). The DNA sample was amplified with a OneTaq Hotstart DNA Polymerase Gradient PCR program ranging from 55°C to 45°C. A separate Gradient ranging from 45.6°C to 40°C was performed of which only the 40°C results are displayed in (M).
  • X axis displays the length of the analyzed fragment in base pairs and Y axis displays the signal strength in RFU.
  • The“sz” value represents the length of the fragment, while “ht” is the peak height in RFU.
  • a peak on the Y axis represents a fluorescence signal strength dependent number of amplicons at its respective length on the X axis.
  • the panels alternate between a 3 LNA reaction and a reaction without LNA, both at the same temperature, as can be seen in the left column.
  • the temperatures in the order of the first two panels representing the highest temperature are 55.0°C(A, B), 53.0°C(C, D), 50.6°C (E, F), 48.2°C (G, H), 45.9°C (I, J), 45°C (K, L).
  • (M) shows an additional reaction at 40°C.
  • the present invention provides a method for the amplification of at least one short tandem repeat (STR) locus in a DNA containing sample.
  • the method can be used for genetic profiling, i.e. for determining genetic fingerprints.
  • the complexity of the fingerprints can be adjusted by the particular primer design, e.g. by the sequence, length and content of uni ver sal/ degenerated bases.
  • the primer(s), which are specific for the STR(s) are "anchored” exactly at the 3’ end of the respective STR, which allows an accurate length measurement, even if the sequence context of the STR is unknown.
  • the reverse primers (herein“second primers”) hybridize outside the respective STR loci to the complementary nucleic acid strain so that the first and second primers together allow for the amplification and analysis (length and/or sequence) of the complete STR sequence.
  • the first and/or second primers comprise one or more locked-nucleic-acid (LNA) nucleotide residues (typically between 3 and 5, but most usually 3 LNA residues).
  • LNA nucleotides confer a considerably increased binding affinity for their template, thus allowing the use of relatively short primers optimized according to statistical calculations and, in particular, the anchoring of otherwise unspecific primer sequences at specific positions in the genome of any - even unknown - species.
  • the present method is, thus, able to provide a single unique assay that can be applied for any source of the STR-containing nucleic acids.
  • a universal assay can be used by all interested institutions including authorities, zoos, breeders, traders and processors of wildlife.
  • the method of the invention it is possible to assign a genetic fingerprint to every individual organism (as long as it has STRs in its genome) as it has long been known in the context of forensics. This procedure therefore has the potential to enforce the regulations of the Washington Convention on International Trade in Endangered Species of Wild Fauna and Flora in a legally secure and economical way.
  • Typical applications of the method of the present invention include the fingerprinting for parentage or lineage analysis or in forensic setting and any use in which individual organisms are to be identified by their genetic fingerprints such as for tracing protected or endangered wildlife.
  • the method of the invention solves these problems by using a semi-statistical, but nevertheless controllable and targeted method which allows the complexity of the data to be adjusted to a selectable size.
  • the method of the invention is based on the fact that small fragments from any sample can be amplified with PCR and virtually every resulting PCR fragment contains a highly informative information carrier (microsatellite, STR) and thus generates a maximum of information with a minimum amount of fragments of minimal size.
  • STR highly informative information carrier
  • PCR primers are used which are anchored at the 3’ end of the STR sequence. Sequences fully complementary to STRs (i.e. without an anchor sequence) are not directly used as PCR primers herein. There are two reasons for this: a primer that hybridizes with a microsatellite can hybridize (prime) at any point on the STR, so that not only one fragment is produced. Hence, it is not possible to determine the exact length of just one precise fragment (the allele of the STR). Moreover, a primer hybridized to anywhere within an STR sequence (i.e.
  • the present invention uses a PCR primer that binds exactly at the beginning in such a way that it always binds exactly and only exactly at the beginning (or just "end") of the microsatellite.
  • a microsatellite is defined by the fact that it has a certain minimum length, which also requires a certain minimum length of the primers used. This forces the use of PCR conditions which can only be specifically constructed by a relatively high temperature. In order to ensure that the primer is "anchored" exactly at the beginning or end of the microsatellite, i.e.
  • a relatively long sequence of nucleotides would have to be added at the beginning (5’ end) the primer which forces the primer to hybridize exactly only at the border between the normal genome and a microsatellite. This sequence would have to be so long that it statistically occurs only extremely rarely in a genome, or be given to react in so many variations of different primers (i.e. a primer mixture) that the sheer amount of primers would make a PCR reaction impossible.
  • second primer An analogous problem exists on the reverse primer side (herein the“second primer”). To base this on homologies between - especially evolutionary very distant species - is practically impossible, since it is so unlikely (because it is statistically hardly ever the case that a homology is in practical proximity to a microsatellite) that such a procedure has no chance of success. Nevertheless, the approach to base the reverse primer on statistically or semi- statistically designed primers is the only possible one.
  • a forward primer (first primer) for this type of microsatellite could look like this in an extreme case:
  • the anchor part of the first primer comprises at least one, "Locked Nucleic Acids” (LNAs) residues. It is also possible that all bases of the anchor are in LNA nucleotides.
  • the bases of these modified LNA nucleotide residues bind just as specifically with their respective complementary base (C with G and A with T) as normal bases but have a much stronger bond with the complementary base.
  • LNA bases the primer contains the greater the affinity for the target (template) strand, up to the point where this is so much greater than that of the STR sequence itself that even unspecific binding of only the anchor would be possible. While this would not lead to exponential amplification (since the 3 '-end of the primer does not fit onto the template strand and thus prolongation would be very unlikely), it could bind such large amounts of LNA (or LNA-containing) anchors non-specifically to the genome that these primers would no longer be available for a specific reaction, especially in later cycles of a PCR. In order to prevent such behavior, it is also possible to incorporate a number of LNA residues into the STR sequence of the primer and thus increase the affinity of this part of the primer for the actual target sequence.
  • the reverse primer (second primer herein) binds to a sequence outside of the microsatellite.
  • a short second primer of 4-8 bases length will bind specifically in any genome with a relatively high frequency depending on its length (longer primers bind less frequently).
  • Such short second primers generates on average according to the rules of normal distribution, together with the first primer a population of fragments whose average length depends on the length of the primers, or on the number of variable positions, or on the number of different primers used with the same or similar properties.
  • more than a single primer can of course be used in an amplification - even at a given length - so that the number of sequences used already provides a very precise (naturally based on the statistical distribution) control of the length of the fragments amplified from a microsatellite together with such a primer.
  • This possibility to vary the degree of specificity, the length and the number of different reverse primers allows to create populations of fragments that are optimized for the final analysis method.
  • the design of the reverse primer according to the above requirements therefore allows the precise optimization of procedures for a wide range of applications, which can range from a simple paternity analysis to the complex determination of the most complicated genetic questions.
  • the second primer is designed so that it binds to the other end of the STR locus on the other strand, i.e. the second primer comprises an anchor sequence and an STR sequence as defined for the first primer.
  • Such a variant method also allows the amplification of fragments which, if two different microsatellite-specific primers with anchors at both ends of the microsatellite are used, are only as short as the microsatellite itself plus the anchor sequences extending beyond it.
  • the possibility of producing such short microsatellite fragments allows for example forensic analyses to be carried out even from materials or samples whose DNA has been degraded to a minimal size.
  • LNA bases in the short primers described here as reverse primers makes it possible to produce a population of fragments based purely on statistical methods, but highly reproducible, which can then be processed by any state-of-the- art method.
  • Prior art RAD techniques are based on restriction digests of entire genomes at specific sites and a genome can then be reproducibly reduced to a certain complexity by fractionation according to the length of the fragments, or only fragments of a certain length range can be selected and then sequenced, e.g. with an NGS technique.
  • the methods of the invention allow exactly the same methodology, but without being restricted by the limited number of possible interfaces for restriction enzymes. It is therefore possible to set the number and average length of the resulting fragments much more precisely, which allows for a variety of applications that are not possible with previous RAD methods.
  • the first primer is designed to introduce a restriction site into the amplified fragment which then serves as the basis of an RAD or ddRAD method for analysis.
  • the restriction cleavage site can be inserted in the middle or near the ends of the primer.
  • the fragment amplified from this first primer can then, after a first amplification step, be subjected to restriction and subsequent ligation of an adaptor and/or barcode sequence.
  • a barcode is artificially used for identification in an NGS sequencing and can be used for a further amplification, which is then performed with only this latter adaptor primer, thus making the advantages of RAD and other methods based on adaptor ligation accessible to the user.
  • mixtures of first primers and/or mixtures of second primers can be used (e.g. degenerate oligonucleotides).
  • microsatellite In the context of the present invention, the terms“microsatellite”,“short tandem repeat” (STR) and“simple sequence repeats” (SSRs) are used synonymously. They relate to a tract of tandemly repeated (i.e. adjacent) DNA motifs that range in length from one to ten, most commonly up to six nucleotides and are typically repeated 5-50 times. Microsatellites can occur at thousands of locations within an organism's genome and have a higher mutation rate than other areas of DNA leading to high genetic diversity. This can be exploited in genetic profiling.
  • STR short tandem repeat
  • SSRs simple sequence repeats
  • the present invention relates to a method for the amplification of at least one short tandem repeat (STR) locus on a double stranded nucleic acid in a sample, the method comprising the steps of
  • amplifying the STR locus in an amplification reaction comprising contacting the sample with a pair for primers, wherein
  • the first primer of said pair of primers comprises a 5’ anchor sequence and a 3’ repeat sequence, wherein
  • the 5’ anchor sequence of said first primer hybridizes under stringent conditions to the nucleic acid sequence immediately downstream of said STR locus on the first strand of said nucleic acid
  • the 3’ repeat sequence of said first primer hybridizes under stringent conditions to the 3’ portion of said STR locus on the first strand of said nucleic acid
  • the present invention relates to a method for the amplification of at least one short tandem repeat (STR) locus on a double stranded nucleic acid in a sample, the method comprising the steps of
  • amplifying the STR locus in an amplification reaction comprising contacting the sample with a pair for primers, wherein
  • the first primer of said pair of primers comprises a 5’ anchor sequence and a 3’ repeat sequence, wherein
  • the 5’ anchor sequence of said first primer is complementary to a nucleic acid sequence immediately downstream of said STR locus on the first strand of said nucleic acid
  • the 3’ repeat sequence of said first primer is complementary to the 3’ portion of said STR locus on the first strand of said nucleic acid
  • the second primer of said pair of primers is complementary to a sequence downstream of said STR locus on the second strand of said nucleic acid.
  • the amplification reaction can be linear or exponential. Most often, the amplification reaction will be a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the first primer has a length of between 12 and 30 nucleotide residues, preferably between 15 and 25 nucleotide residues, even more preferably between 18 and 21 nucleotide residues, most preferably the first primer has a length of 18 nucleotide residues.
  • the second primer has typically a length of between 12 and 30 nucleotide residues, preferably between 18 and 25 nucleotide residues, most preferably between 20 and 24 nucleotide residues; it can, however, be much shorter as outlined herein below.
  • the 5’ anchor sequence of the first primer has typically a length of between 4 and 10 nucleotide residues, preferably between 5 and 7 nucleotide residues, most preferably 6 nucleotide residues.
  • the 3’ repeat sequence of the first primer has typically a length of between 8 and 16 nucleotide residues, preferably between 10 and 14 nucleotide residues, most preferably 12 nucleotide residues.
  • the 5’ anchor sequence is immediately adjacent to the 3’ repeat sequence. Hence, it is possible that the sequence of the first primer consists of the 5’ anchor sequence and the 3’ repeat sequence.
  • the primers may comprise one or more non-naturally occurring nucleotides that have an increased hybridization property towards the nucleotide position to which they are supposed to pair.
  • Most prominent examples for such non-naturally occurring nucleotides are locked nucleic acid (LNA) nucleotides.
  • LNA nucleotide is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the 3'-endo (North) conformation.
  • LNA nucleotides hybridize with DNA according to Watson-Crick base-pairing rules.
  • the first and/or the second primer comprises one or more modified bases, preferably the first and/or second primer comprises one or more locked-nucleic-acid (LNA) nucleotide residues.
  • the first primer comprises between 2 and 6 LNA nucleotide residues, preferably between 3 and 5 LNA nucleotide residues, most preferably 3 LNA nucleotide residues.
  • the 5’ anchor sequence of the first primer may in particular comprise between 1 and 4 LNA nucleotide residues, preferably 2 or 3 LNA nucleotide residues.
  • the 3’ repeat sequence of the first primer may in particular comprise between 1 and 4 LNA nucleotide residues, preferably 1 or 2 LNA nucleotide residues.
  • the second primer may in particular comprise between 2 and 6 LNA nucleotide residues, preferably between 3 and 5 LNA nucleotide residues, most preferably 3 LNA nucleotide residues.
  • the first and/or second primers may also comprise a covalently attached minor groove binder (MGB), preferably wherein the MGB is conjugated to the 3’ end of the primer.
  • MGB minor groove binder
  • A“Minor- Groove-Binder“ (MGB) herein is for example dihydropyrroloindole carboxylate tripeptide (CDPL).
  • CDPL dihydropyrroloindole carboxylate tripeptide
  • attached MGBs can increase the affinity of the primer for the target sequence, thus allowing shorter primer sequences.
  • a second primer which has a length of between 4 and 12, preferably between 8 and 10 nucleotide residues, is possible. The shorter the second primer the more universal it will be.
  • the method of the invention may comprise the step of isolating the nucleic acid from the sample prior to the amplification reaction. It may also comprise as step of a step of processing the sample prior to the amplification reaction, e.g. purifying and/or concentrating the nucleic acid.
  • two or more STR loci can in some aspects be amplified, e.g. by contacting the sample with a distinct second primer for each STR locus to be amplified.
  • a distinct second primer for each STR locus to be amplified For example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 STR loci are amplified in parallel.
  • Some typical forensic applications for example include between 9 or 13 STR loci.
  • the sample may be any sample which contains nucleic acids, typically DNA, having microsatellite sequences.
  • the sample may, thus, e.g. be a forensic sample (e.g. from a crime scene) or a biological sample taken from an animal or plant.
  • the biological sample may be any kind of DNA containing sample, e.g. a fluid such as blood, plasma, serum or a tissue sample.
  • the method of the invention typically comprises determined the length and/or sequence of the amplified fragment or fragments. Sequencing methods such as next generation sequencing methods can be employed, such as Illumina (Solexa) sequencing, Roche 454 sequencing, ABI SOLiD sequencing, Ion Torrent proton sequencing (e.g. Thermo Fisher), and Nanopore Sequencing.
  • next generation sequencing methods such as Illumina (Solexa) sequencing, Roche 454 sequencing, ABI SOLiD sequencing, Ion Torrent proton sequencing (e.g. Thermo Fisher), and Nanopore Sequencing.
  • Length determination of the amplified fragment(s) can for example be achieved by electrophoretic or chromatographic methods such as (agarose) gel electrophoresis or high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC).
  • electrophoretic or chromatographic methods such as (agarose) gel electrophoresis or high performance liquid chromatography (HPLC) or ultra high performance liquid chromatography (UHPLC).
  • the first primer will be complementary to a sequence spanning the 3’ end of an STR locus and the flanking sequence 3’ thereof.
  • the first primer may comprise degenerate bases such as inosine, particularly in the 5’ anchor sequence in order to allow hybridization with multiple STR loci. To a certain degree mismatches may be tolerated in some instances by using less base-pair-specific nucleoside analogues.
  • multiple second primers (each specific for a particular STR locus) can be used in combination with one or more first primers.
  • the second primer can also be a mixture of multiple primers, e.g. which comprise degenerate bases such as inosine and/or which have random sequences.
  • the second primer can be relatively short, e.g. 4 to 10 nucleotides in length. adapting the length and/or degree of degeneracy allows for adjusting the (statistically expected) length distribution of the amplified fragments.
  • the first and/or second primers may contain a covalently attached dye such as FAM, HEX, TET, JOE, VIC, NED, PET, ROX, TAMRA or Texas Red®. This for example allows for a detection of the amplification reaction in real time-PCR.
  • the first and/or second primer may also be covalently linked to a capture moiety such as biotin which allows for the selective extraction of (a) particular amplified fragment(s) from a complex mixture of amplified fragments.
  • a capture moiety such as biotin which allows for the selective extraction of (a) particular amplified fragment(s) from a complex mixture of amplified fragments.
  • the first and/or second primers may also comprise barcode sequences and/or adaptor sequences in order to identify individual amplified fragments.
  • the first and/or second primers may in addition comprise a further barcode which identifies the sample from which the STR sequence(s) has/have been amplified.
  • Molecular barcode technologies are available, through any of the commercial suppliers of NGS -technology but can also be self-designed and ordered by many oligonucleotide firms.
  • the first and/or second primer may also additionally comprise a sequence which introduces a restriction site into the amplified fragment(s).
  • Stringent hybridization conditions as used herein are for example described in Sambrook et ah, Molecular Cloning, A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • An example of stringent hybridization conditions is the hybridization in 50% formamide, 5> ⁇ SSC, 5x Denhardfs solution, 40 mM sodium phosphate pH 6.8; 0.5% (wt./vol.) BSA, 1% (wt./vol.) SDS, 0.1 mg/ml herring sperm DNA at a hybridization temperature of 42° C and subsequently washing the filters in 0.5> ⁇ SSC/0.5% SDS at 60° C.
  • the amplification reaction in particular, PCR, can also be a multi-step process in which a pre-amplification is performed followed by a purification which is again followed by another amplification reaction.
  • the first primer can be labelled with a capture molecule in the first amplification reaction and with another label such as a barcode sequence in the second amplification reaction.
  • the amplification method of the present invention and the primers described herein can be used for genetic profiling (or DNA fingerprinting). Genetic profiling can for example be used for forensic purposes, e.g. of DNA-containing samples derived from crime scenes or in the context of criminal investigations.
  • STRs genes which comprise STRs in their genome
  • They can also be employed for determining genetic (family) relationships between organisms which comprise STRs in their genome such as plants, human and non-human animals. They can be used in the context of kinship analysis, e.g. for paternity, maternity, siblingship testing.
  • the present methods and their results can also more widely be used in the context of genetic fingerprinting.
  • These genetic fingerprints can be used for identifying individual organisms and these genetic fingerprints can be stored in databases and can be made available for comparisons or identification purposes. This is for example relevant in the context of the traceability or heritage analysis of individual animals or plants, e.g. of endangered, protected and/or rare animal or plant species or subspecies.
  • individuals of protected species can be accompanied by an ID document determined using the methods of the invention or the primers described herein. This is, e.g., relevant when such animals are transferred between zoos, breeders, collectors or other institutions, or for the purposes of monitoring or law enforcement authorities.
  • By comparing an individual’s genetic fingerprint with a database the identity of that subject can be verified. In this sense, each individual tested can be assigned a unique“address” based on its genetic fingerprint.
  • the genetic fingerprint can for example be saved and/or processed in a dedicated data format, e.g. using block chain technology in order to identify or trace a particular individual. For this purpose, e.g. a “hash” value of a genetic fingerprint can be calculated that allows unambiguous identification but no back-calculation of the original genetic fingerprint. It is important that the data format used is protected against counterfeit.
  • the genetic fingerprints or their hash values can be included in breed registries, herdbooks, studbooks or other relevant registers. Such registers can be kept electronically and may be based on information stored and/or processed using block chain technology.
  • the present invention is of particular use in enforcing the provisions of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), also known as the Washington Convention.
  • the present invention in particular aspects relates to the following items:
  • a primer with an anchor according to item 1) which does not consist of only one specific sequence, but contains variable bases at some positions (according to IUPAC code or also a combination of two specific primers which hybridize in combination in the same reaction at the relevant position with several sequence variants), which bind to more than one site in the genome of any living being and thus allow the simultaneous amplification of at least two, often the number of fragments of internationally standardized genetic fingerprints, but in our preferred variants several tens or even hundreds of STRs with the target sequence in the same reaction.
  • Reverse primer consisting of a sequence unique in the genome, which, together with a primer anchored to an STR, amplifies a specific fragment, the length of which can then be measured, or the sequence between the primers can additionally be determined by any DNA sequence method.
  • a reverse primer which according to items 1) and 2) does not consist of a specific sequence, but similar to the anchor consists of a mixture of many relatively short primers or a sequence which contains sequences which are variable between one and several positions according to IUPAC code and thus binds not only at one but at several or many positions in the genome and thus in combination with forward primers according to items 1) and 2) amplifies more than only one STR in such a way that a reliable length measurement and also a determination of the sequence between the primers is possible.
  • a mixture of reverse primers according to item 4), which are varied in their length and by this possible exact adjustment of the length of the primer can adjust the statistical frequency of the binding of the primer in the genome and thus the average length of the resulting fragments can be precisely adjusted.
  • a primer which consists of bases (or naturally occurring bases with analogous properties) modified in this way at at least one, but at most all, of its base positions, which primer contains a binding affinity for the respective base to be paired in the template strand which is substantially increased compared to a normal base pairing.
  • One or more bases built into Forward or Reverse primers which can be chemical modification or naturally occurring base with analogous properties, which unspecifically pairs with either all other bases or at least more than a single base and thus allows the construction of a primer which ignores the lack of complementarity at the respective positions of these particular bases, so to speak, and thus allows the construction of primers, which are insensitive to mismatches (non-homologous base positions between primer and template strand) and thus allow variability of the binding site of the primer(s) without having to use multiple primers different at the site in question or primers degenerated according to IUPAC code.
  • a primer according to the above items which, in order to increase the binding affinity of the statistically forced anchor or very short reverse primer for amplification of multiple fragments at the 5' end of the primer, has a chemical molecule which, like a so-called “minor groove binder", has a similar effect on the binding affinity of a very short sequence as primer.
  • each forward primer carries a different marker or one of both or both primers carry several different markers, e.g. a color marker, which, by detecting the combination of e.g. colors, allows an amplified fragment to be identified even in a complex mixture.
  • markers e.g. a color marker
  • a forward primer according to the above items which carries an additional or even only one label, such as biotin or a similar molecule, which allows to extract only those amplified fragments from a complex mixture of amplificates which actually contain the STR sequences sought and with which it is possible to remove amplifications between the reverse primers from such a complex mixture.
  • an additional or even only one label such as biotin or a similar molecule
  • a forward primer which carries 5' from the anchor a bridging sequence which most likely does not hybridize with any sequence preceding the anchor and to which a sequence barcode is attached which identifies the exact fragment amplified by the primer or even a small subset of these amplicons by its sequence unique to each forward primer and can serve as address/identifier for each amplified fragment.
  • a reverse primer which, if a forward primer with a specific sequence barcode cannot uniquely identify a fragment, a short sequence barcode attached directly or via a sequence bridge is also able to provide, together with the barcode of the forward primer, this precise unique identification of the fragment for exactly one fragment.
  • a forward primer to which a further barcode is attached for the purpose of carrying out a large number of experiments from a large number of individuals simultaneously, which, as is already common practice today in the NGS library creation, does not precisely identify the fragment but the origin of the analysed sample.
  • a primer which according to item 11) comprises the further steps from items 12) to 16).
  • An address system resulting from the above items which comprises the methods in items 11) to 17) for any living being which has STRs in its genome, and which therefore makes possible for any existing species of living being containing STRs, i.e. also for any individual/species of a species, an address system reproducible for this and closely related species, i.e. allows any genetically solvable questions within a species by means of a reproducibly comparable database, but also allows species and subspecies and other determinations between relatively closely related species.
  • a two-step amplification in which first a pre-amplification with a small number of PCR cycles is carried out with forward primers which are labelled with a capture molecule and serve to purify the reaction from such fragments before a second amplification step.
  • a CITES control system e.g. a block chain, or a transaction management system based on a similar forgery-proof coding of such a genetic fingerprint for all CITES or nationally or regionally held, bred, traded or processed organisms or products derived from them.
  • a reverse primer attached to randomly fragmented DNA resulting in fragments large enough to contain an STRs and sufficient downstream sequence to allow PCR by virtue of attaching a universal PCR-priming adaptor to the randomly designed DNA-ends and PCR with the anchor as forward and the adaptor-primer as the reverse, which is the same for all fragments in the fractionated mixture.
  • Figure 1 illustrates the primer design as disclosed herein and used in the context of the method of the present invention.
  • the method relates to the amplification of short tandem repeat (STR) locus comprising a first primer having a 5’ anchor sequence and a 3’ repeat sequence and a second primer hybridizing downstream of said STR locus.
  • STR short tandem repeat
  • Samples of a known genotype were verified using the PowerPlex® 21 System (PP21, Promega), a multiplex STR system for human identification analysis including forensic analysis. Primers were designed to detect three well established human STR loci, i.e. loci D10S1248, D12S391, SE33.
  • FIG 2 an electropherogram of genotype of Individual 1 verified by PP21 is exemplarily shown.
  • the marker D10S1248 is comprised in alleles 14 and 16 (D10S1248 14/16) and the marker SE33 is comprised in alleles 17 and 20 (SE33 17/20).
  • the marker D12S391 is comprised in alleles 18 and 20 (D12S391 18/22).
  • Primers were designed using the published sequences for each locus. As a rule, the 6-7 bases preceding the polymorphic STR locus were used, followed by 12 bases (3 repeat motifs for a tetranucleotide repeat STR) of polymorphic sequence. Reverse primers were chosen via usual primer design criteria (GC rich region, roughly 20 bp long) and chosen to be as close as possible to the 3’ end of the polymorphic STR sequence.
  • the DNA sample of the genotype D10S1248 14/16 was amplified using a OneTaq Hotstart DNA Polymerase (NEB) protocol in a 12.5 pL reaction that contained per reaction (4 in total) 5.6pL of nuclease free water 2.5 pL of 5x OneTaq standard Buffer, 2pL dNTP’s (1.25mM), 0.2 pL of OneTaq DNA Polymerase and 0.6 pL of forward ) and 0.6 pL reverse Primers (10 pM) (Microsynth AG).
  • NEB OneTaq Hotstart DNA Polymerase
  • the gradient reactions were performed in a Tgradient PCR machine (Biometra), at two different temperatures, each covering both versions of the LNA modified primers (3 LNA bases and 5 LNA bases). The temperatures were 52°C, 58°C and all forward primers were fluorescence labeled at 5’ end with a FAM fluorescein dye.
  • primer sequences and internal LNA placements see, e.g., Table 1, Table 2 and Fig. 4.1.
  • Capillary electrophoresis was performed using an ABI Prism 310 with POP7 polymer.
  • 1 pL of PCR product was diluted 1 :4 with nuclease free water and of this mix 1 pL was added to 12 pL of a 1 :24 size standard (LSRox 200): HiDi solution.
  • the injection was set to a duration of 3 seconds, 15.0 kV injection voltage.
  • the run was performed at 60°C and 15.0 kV for a total of 21 minutes.
  • the PCR product was diluted 1 : 10 with nuclease free water to reduce the resolution of the signal of the undiluted PCR product.
  • DNA sample of individual 1 who showed the genotype SE33 17/20 (see, e.g., Figure 2) for this marker was amplified using a OneTaq Hotstart DNA Polymerase (NEB) protocol in a 12.5 pL reaction that contained per reaction (8 in total) 5.6pL of nuclease free water 2.5pL of 5x OneTaq standard Buffer, 2qL dNTP’s (1.25mM), 0.2 qL of OneTaq DNA Polymerase and 0.6 qL of forward and 0.6 qL reverse Primers (10 qM) (Microsynth AG).
  • NEB OneTaq Hotstart DNA Polymerase
  • the gradient reactions were performed in a Tgradient PCR machine (Biometra), at four different temperatures, each covering both versions of the LNA modified primers (3 LNA bases and 5 LNA bases). The temperatures were 55.6°C, 56.8°C, 58.0°C, 59.1°C and all forward primers were fluorescence labeled at 5’ end with a FAM fluorescein dye.
  • Tgradient PCR machine Biometra
  • Capillary electrophoresis was performed using an ABI Prism 310 with POP7 polymer.
  • 1 pL of PCR product was diluted 1 :4 with nuclease free water and of this mix 1 qL was added to 12 qL of a 1 :24 size standard (LSRox 500): HiDi solution.
  • the injection was set to a duration of 1 second and the injection voltage was set to 15.0 kV.
  • the run was performed at 60°C and 15.0 kV for a total of 21 minutes.
  • DNA samples of 8 individuals were isolated using standard Qiagen DNA-extractions kits (see, e.g., Fig. 2 for respective genotypes) and amplified using a OneTaq Hotstart DNA Polymerase (NEB) protocol in a 12.5qL reaction, using the previously described parameters with the only difference being primer concentration and consequently water volume.
  • NEB OneTaq Hotstart DNA Polymerase
  • 0.25 qL of each Forward and reverse primer (IOmM) for the markers D10S1248 (or denoted as marker D10), (D12S391 or denoted as marker D12) and SE33 that each contained 3 internal LNA bases (for exact positions see Table 1 and 2 as well as Fig. 4.1) were added, and the water volume was adjusted to 5.3 qL.
  • Capillary electrophoresis was performed using an ABI Prism 310 with POP7 polymer.
  • 1 qL of PCR product was diluted 1 :4 with nuclease free water and of this mix 1 qL was added to 12 qL of a 1 :24, size standard (LSRox 500): HiDi, solution.
  • the injection was set to a duration of 3 seconds, 15.0 kV injection voltage.
  • the run was performed at 60°C and 15.0 kV for a total of 21 minutes.
  • the modified D10 Primer sequence (see Table 1) was tested without internal LNA bases (SEQ ID NO.5).
  • the previously described PCR protocol was used (see materials and methods corresponding to the D10S1248 experiments).
  • the reactions were performed at a gradient that was set to 55.0°C, 53.0°C, 50.6°C, 48.2°C, 45.9°C, 45°C, with each temperature covering two reactions, one containing a forward primer with 3 internal LNA bases and one reaction in which the forward primer was not fitted with internal LNA bases.
  • An additional PCR was performed with temperatures ranging from 45°C to 40°C of which only the results of the 40°C reaction are displayed.
  • the fragment analysis method on the ABI Prims 310 was the same as in the previous experiment.
  • the detection of the D10 marker was assessed by amplifying an STR locus comprising said marker using either the forward primer 1 or 2 (“anchored primers”) and the reverse primers as shown in Table 1.
  • Each of the two forward primers as shown in Table 1 are examples for a first primer as used in context of the method disclosed herein and in the context of the invention, i.e. a first primer comprising a 5’ anchor sequence and a 3’ repeat sequence.
  • Both forward primers as shown in Table 1 are 18 bp long.
  • the 6 bp at the 5’ end (indicated in Table 1 in italic) of the forward primer correspond to the 5’ anchor sequence of said first primer, i.e.
  • the residual 12 bp (indicated in bold in Table 1) of the forward primer correspond to the 3’ repeat sequence of said first primer which hybridizes to the 3’ portion of the STR locus (in here 3 repeat motifs of a tetranucleotide repeat STR) with the microsatellite repeat motif: GGAA.
  • the structural details of the two anchored primers tested are shown in Table 1.
  • Forward primer 1 contains 5 LNA bases (3 in the 5' anchor sequence and 2 in the 3' repeat sequence; see bases marked in grey in first line of Table 1) and forward primer 2 contains 3 LNA bases (2 in the 5' anchor sequence and 1 in the 3' repeat sequence; see bases marked in grey in second line of Table 1), respectively.
  • Table 1 Forward primers 1 and 2 and Reverse primer. Italic bases of forward primers 1 and 2 represent the anchor, bold bases represent the STR repeat motif and grey bases represent
  • the PCR product was diluted 1 : 10 with nuclease free water and capillary electrophoresis was performed and analyzed using an ABI Prism 310 series.
  • Figure 3 shows four electropherograms of the anchor modified D10 assay, i.e. an assay which was performed as described in the section “D10S1248 experiments” herein above and which was modified by using either forward primer 1 having 5 LNA bases (two electropherograms on the left) or forward primer 2 having 3 LNA bases (two electropherograms on the right) and the reverse primer as shown in Table 1 for the amplification reaction.
  • the amplification was carried out at two different temperatures, i.e. at 58 °C (upper two electropherograms) and 52°C (two lower electropherograms).
  • the X axis of Figure 3 displays the length of the analyzed fragment/amplicon in base pairs and the Y axis displays the signal strength in relative fluorescence units (RFU).
  • the height of the peaks as shown in Figure 3 directly correspond to the strength of a fluorescence signal which indicates the number of fragments/amplicons with a particular length as shown on the X axis.
  • the detection of the SE33 marker was assessed by amplifying an STR locus comprising said marker using either the forward primer 3 or 4 (“anchored primers”) and the reverse primers as shown in Table 2. Particularly, to test whether the anchored primers can be applied to all circumstances that would be required by a forensic test, a microsatellite locus that differs from the D10S1248 locus in a way that is fairly common for STR’s was targeted.
  • the SE33 locus unlike D10S1248, has a flanking region which contains degenerate repeat motifs of a sequence similar to that of the polymorphic site.
  • a similar primer design as with the D10S1248 marker was used, with the only difference being, that it anchored itself into a suitable position within the degenerate flanking region of the STR.
  • Figure 4.1 displays a sequence (SEQ ID NO.9) comprising the polymorphic STR region (light grey) (SEQ ID NO.10) and the 5’ flanking sequence (white) (SEQ ID NO. i l) containing an anchored primer (dark grey) (SEQ ID NO.12).
  • Table 2 Forward primers 3 and 4 and Reverse primer. Italic bases of forward primers 3 and 4 represent the anchor, bold bases represent the STR repeat motif and grey bases represent LNA.
  • the experiment was performed as described herein above in the section“SE33 experiments”. Particularly, a DNA sample was taken from Individual 1 (see Figure 2 for the corresponding genotype SE33 17/20). The DNA sample of Individual 1 was amplified using a OneTaq Hotstart DNA Polymerase (NEB) protocol by using either forward primer 3 or 4 and the reverse primer as shown in Table 2. The PCR product was diluted 1 :4 with nuclease free water and capillary electrophoresis was performed and analyzed using an ABI Prism 310 series.
  • NEB OneTaq Hotstart DNA Polymerase
  • Figure 4.2 shows four electropherograms of the anchor modified SE33 assay, i.e. an assay which was performed as described in the section“SE33 experiments” herein above and which was modified by using either forward primer 3 having 5 LNA bases (electropherograms on the left) or forward primer 2 having 3 LNA bases (electropherograms on the right) and the reverse primer as shown in Table 2 for the amplification reaction.
  • the amplification was carried out at two different temperatures as indicated on the left of Figure 4.2.
  • the X axis of Figure 4.2 displays the length of the analyzed fragment/amplicon in base pairs and the Y axis displays the signal strength in relative fluorescence units (RFU).
  • the height of the peaks as shown in Figure 4.2 directly correspond to the strength of a fluorescence signal which indicates the number of fragments/amplicons with a particular length as shown on the X axis.
  • the resulting Peak pattern as shown in the electropherograms of Figure 4.2 allowed deduction of the correct genotype, confirmed with the PP21 genotyping kit.
  • Amplicons as shown by the electropherograms in Figure 4.2 were also very specific at all temperatures and LNA-Primer constellations tested. Primers containing 5 LNA bases showed higher peak stutter when compared to ones containing 3 LNA bases, and the signals themselves were stronger for 5 LNA primers.
  • the correct genotypes could be produced by the individual markers (i.e. D10S1248 and SE33 marker) assessed.
  • these markers were tested in a multiplexed reaction.
  • Three primer pairs of the previously verified markers D10S1248, (D12S391), and SE33, containing 3-LNA bases in the forward primer were fused into a multiplex assay at an annealing temperature of 58°C.
  • the forward primers comprising 3-LNA bases were chosen.
  • the forward primers were all labeled with a FAM fluorescein Dye and the potential fragment length for each allele of every marker was considered and adjusted accordingly. The results are shown in Figure 5 A for individual 1 and in Figure 5 B for individual 2.
  • the electropherograms in Figures 5 A and B show that the fragment lengths correspond to the genotype verified by the PP21 kit (see, e.g., Fig. 2). It was observed that the SE33 signal was only slightly weaker than the signal of D10S1248 and D12S391.
  • the forward primers with LNA bases bind the 5’ anchor with a higher specificity than the 3’ STR motif which has multiple hybridization sites in the same microsatellite.
  • the effect of the LNA containing primers in comparison to ones with the same DNA sequence without LNA was tested. Since LNA bases are supposed to influence specificity as well as the Tm value of the primers, their effect using a gradient PCR reaction was verified. D10S1248 marker was used for this experiment because its forward primer has the shortest anchor sequence. Since LNA bases influence specificity, a set of primers that score low in this discipline are the best model to observe their effect.
  • Degraded DNA samples are one of the biggest challenges faced by forensic biologists when trying to determine Genotypes. Amplified degraded samples produce results that are incomplete and can undermine an investigation of a suspect because of potentially misleading information. As illustrated by the appended Examples above, the method as described herein and in the context of the invention provides a solution to this problem, because the range of amplicons it produces is as short, or shorter than currently available commercial STR kits (see, e.g., Butler et al. 2003). The markers also do not have to be limited to one per dye because most markers are still very short even when they are adjusted in size to compensate for shorter markers within the same dye and are consequently not as vulnerable to degraded samples.
  • an amplification method using a forward primer as described herein and in the context of the invention delivers a proof of principle and fundamental information for this method of STR amplification to be feasible and applicable in other fields of molecular genetics that require short amplicons of specific STR. In this context this method could also be adjusted to other applications. If one were to shorten the base pair sequence preceding the Primer internal STR motif by only one or two base pairs, the primer is no longer locus specific and will amplify throughout the entire genome every locus STR locus with this flanking sequence.

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Abstract

La présente invention concerne un procédé d'amplification d'au moins un locus de répétitions courtes en tandem (STR) sur un acide nucléique double brin dans un échantillon. Ledit procédé utilise une première amorce de ladite paire d'amorces comprenant un séquence d'ancrage (5') et une séquence de répétition (3') et une seconde amorce s'hybridant, dans des conditions stringentes, à une séquence en aval dudit locus STR sur le second brin dudit acide nucléique. Le procédé peut être utilisé pour le profilage génétique, par exemple à des fins médico-légales ou dans le contexte de la protection de la faune sauvage.
PCT/EP2020/071050 2019-07-26 2020-07-24 Procédé de profilage génétique WO2021018802A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001088189A2 (fr) * 2000-05-15 2001-11-22 Keygene N.V. Technique d'empreinte d'adn aflp$m(3) basee sur des microsatellites
WO2009059049A1 (fr) * 2007-10-30 2009-05-07 Aplied Biosystems Inc. Procédés et kits pour l'amplification multiplexée de loci de séquences courtes répétées en tandem
WO2011066467A2 (fr) * 2009-11-25 2011-06-03 Life Technologies Corporation Loci de l'échelle allélique
WO2011097503A2 (fr) * 2010-02-05 2011-08-11 Quest Diagnostics Investments Incorporated Procédé de détection de motifs de séquences répétées dans un acide nucléique
WO2012155084A1 (fr) * 2011-05-12 2012-11-15 Netbio, Inc. Procédés et compositions destinés à l'amplification multiplex rapide de loci de séquences courtes répétées en tandem

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001088189A2 (fr) * 2000-05-15 2001-11-22 Keygene N.V. Technique d'empreinte d'adn aflp$m(3) basee sur des microsatellites
WO2009059049A1 (fr) * 2007-10-30 2009-05-07 Aplied Biosystems Inc. Procédés et kits pour l'amplification multiplexée de loci de séquences courtes répétées en tandem
WO2011066467A2 (fr) * 2009-11-25 2011-06-03 Life Technologies Corporation Loci de l'échelle allélique
WO2011097503A2 (fr) * 2010-02-05 2011-08-11 Quest Diagnostics Investments Incorporated Procédé de détection de motifs de séquences répétées dans un acide nucléique
WO2012155084A1 (fr) * 2011-05-12 2012-11-15 Netbio, Inc. Procédés et compositions destinés à l'amplification multiplex rapide de loci de séquences courtes répétées en tandem

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS

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