EP1853723A1 - Procédé pour déterminer le type d'un individu au moyen de loci de microsatellites (str) de l'adn génomique - Google Patents

Procédé pour déterminer le type d'un individu au moyen de loci de microsatellites (str) de l'adn génomique

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Publication number
EP1853723A1
EP1853723A1 EP06707244A EP06707244A EP1853723A1 EP 1853723 A1 EP1853723 A1 EP 1853723A1 EP 06707244 A EP06707244 A EP 06707244A EP 06707244 A EP06707244 A EP 06707244A EP 1853723 A1 EP1853723 A1 EP 1853723A1
Authority
EP
European Patent Office
Prior art keywords
typing
str
dna
individual
individual according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06707244A
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German (de)
English (en)
Inventor
Klaus Bender
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johannes Gutenberg Universitaet Mainz
Original Assignee
Johannes Gutenberg Universitaet Mainz
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johannes Gutenberg Universitaet Mainz filed Critical Johannes Gutenberg Universitaet Mainz
Publication of EP1853723A1 publication Critical patent/EP1853723A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a novel STR typing strategy which allows the simultaneous amplification and subsequent analysis of multiple (e.g., eleven) polymorphic systems with amplicon sizes less than 270 bp. After PCR amplification, the multiplex reaction is divided into two sets of STR multiplexes and analyzed separately. This multiplex system has been specifically designed and tested for use in forensic investigations when limited amounts or degraded DNA is available, for example, when isolated from telogen hair roots. The present invention also relates to a corresponding kit for STR typing.
  • STR Short Tandem Repeat
  • the primers on the DNA sequence are moved closer to the repeat unit, so that overall shorter DNA fragments can be analyzed.
  • the minimum length of these fragments is 60 to 250 bp [P. Grubwieser, R. Muhlmann, W Parson, New sensitive amplification primers for the STR locus D2S1338 for degraded casework DNA, Int J Legal Med 117 (2003) 185-188; JM Butler, Y. Shen, BR McCord, The development of reduced size STR amplicons as tools for analysis of degraded DNA, J Forensic. 48 (2003) 1054-1064; P. Wiegand, M.
  • this object of the present invention is achieved by a method of typing an individual comprising the steps of a) providing genomic DNA from the individual, b) amplifying at least two short tandem repeat (STR) - Loci of the genomic DNA by means of optionally labeled pairs of amplification primers, at least one primer being provided with a linking group, c) separation of the amplified STR fragments by means of the linking group into at least two fractions of amplicons, and d) separate detection of the STR Fragments of the fractions.
  • STR short tandem repeat
  • Methods thus include the use of binding group (e.g., biotinylated) PCR primers on a subset of the multiplexing reaction. This allows the amplification of overlapping PCR fragments that could not be screened together on capillary electrophoresis without separation.
  • binding group e.g., biotinylated
  • the invention makes it possible to determine as many DNA features as possible from even the smallest DNA traces in one batch. As a result, as little as possible trace material is used to allow a second independent analysis or at least DNA levels, if the entire isolated DNA must be used to be able to determine several characteristics at all. In comparison to successive amplification with solid phase PCR after e.g. Hellmann et al. In addition, there is a significant time saving and a significantly lower risk of contamination. Compared to other multiplex reactions, a significant increase in the number of detectable DNA features is possible.
  • Preferred is a method of typing an individual according to the invention, wherein the subject is a mammal, such as a human.
  • a method of typing an individual according to the invention wherein the STR loci are selected from the group comprising D3S1358, D8S1179, D21S11, THO1, FGA, VWA, D2S1338, D12S391, TPOX, D5S818, D18S51, FES and Amelogenin.
  • the amplification can take place by means of PCR and / or multiplex amplification.
  • the fluorescent dye may be any suitable dye, in particular it is selected from the group comprising 6-FAM, JOE, NED and PET (red).
  • binding group is selected from the group comprising biotin, streptavidin, a His-tag, heat-stable antigen and oligonucleotide.
  • all binding groups can be used which do not interfere with the amplification and are suitable for a subsequent separation of the fractions.
  • This separation of the amplified STR fragments by means of the linking group can comprise a solid phase on which e.g. Biotin, streptavidin, antibodies or complementary oligonucleotides are immobilized
  • the solid phase may comprise a membrane, Sepharose beads or magnetic Sepharose beads. Suitable other phases are well known to those skilled in the art.
  • genomic DNA is derived from blood, blood components, semen and / or telogen hair.
  • the detection of the STR fragments of the fractions comprises a length determination of the fragments, for example by means of capillary gel electrophoresis. Suitable other detection methods are also well known to those skilled in the art.
  • an inventive method for typing an individual wherein as primer pairs at least two pairs selected from the group of SEQ ID Nos. 1-3; 4 and 5; 6 and 7; 8 and 9; 10 and 11; 12 and 13; 14 and 15; 16 and 17; 18 and 19; 20 and 21; and 22 and 23 (see Table 1).
  • Another aspect of the present invention then relates to a method of typing an individual further comprising identifying the individual from the detected STR fragments.
  • Another aspect of the present invention then relates to a diagnostic kit comprising at least two pairs of STR primers for carrying out the method as above, optionally with other materials and excipients.
  • Corresponding materials and adjuvants include, for example, PCR reagents, buffers, solid phases, instructions for use, dyes, and others.
  • a final aspect of the present invention then relates to the use of the method as above or the kit as above in the forensic field.
  • the multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THOI, FGA and VWA), as well as four additional STR systems selected for their robustness (D2S1338, D12S391 , TPOX and D5S818), together with the sex-specific locus amelogenin.
  • ESS European standard set of loci
  • additional STR systems selected for their robustness D2S1338, D12S391 , TPOX and D5S818), together with the sex-specific locus amelogenin.
  • the multiplex reaction is split into two sets of STR multiplexes using biotin-labeled primers for only one set.
  • streptavidin-coated sepharose beads e.g. five STR systems (or even between two and nine) are separated from the remaining (e.g., six) systems before being analyzed in two different runs on a capillary gel electrophoresis instrument.
  • This multiplex system has been specifically designed and tested for use in forensic investigations when limited amounts or highly degraded DNA are available, for example, when isolated from petal hair roots.
  • the inventors describe a method wherein, for example, two 5-plex and 6-plex PCR reactions are combined into a large multiplexing reaction. 10 STR systems plus amelogenin are co-amplified, with maximum fragment sizes up to 262 base pairs (Table 2), and then split into the two initial small multiplex reactions.
  • the primer sequences were selected from published data [P. Grubwie- Ser, R. Muhlmann, W Parson, New Sensitive Amplification primers for the STR locus D2S1338 for degraded casework DNA, Int J Legal Med 117 (2003) 185-188; Y. Shigeta, Y. Yamamoto, Y. Doi, S. Miyaishi, H.
  • the present innovative approach simultaneously combines the multiplex amplification of, for example, eleven loci with very short amplicons with the biochemical separation of the amplicons into eg two fractions of fragments for subsequent electrophoretic analysis.
  • biotin separation process for example, does a little more work and takes time, it has the major advantage of sparing valuable sample material by allowing the simultaneous analysis of ten short Amplikon STR systems from a single DNA aliquot. These methods can be used in cases where most of the conventionally used multiplex STR kits are incapable of producing reliable results.
  • the multiplex includes six of the seven European STR loci for national DNA databases and eight STR loci from the US CODIS database [PD Martin, H.
  • FIG. 1 The allelic ladders for the new Bioplex-11 were reamplified from the allelic standards of the SGM Plus TM (Applied Biosystems) or PowerPlex® 16 (Promega) kits and the "self-assembled" D12S391 ladder.
  • the three top panels represent the two 6-FAM labeled STR systems D3S1358 and D2S1338 along with amelogenin, the two JOE-labeled STR systems D8S1179 and D21S11, as well as the D12S391 system belonging to the 6-plex part of the multiplex.
  • the three lower panels show the two 6-FAM-labeled STR systems THOl and FGA, the two JOE-labeled STR system TPOX and VWA, and the D5S818 system from the biotinylated 5-plex submultiplex.
  • FIG. 1 SENSITY ASSESSMENT Using sequential dilutions of genomic DNA from 500 pg down to 6.25 pg.
  • the screenshot shows only the electrophorogram from the 5-plex part of the multiplex. PCR products were separated and detected on the ABI PRISM 310 Genetic Analyzer. Arrows indicate peaks indicating N and N + 1 fragments that are typically for overamplification of DNA probes.
  • Table 1 PCR primer sequences for amelogenin and STR systems. Underlined are C-stretch sequences for extension of the PCR product. The reference sequences for the STR markers were obtained from the GenBank® (http://cstl.nist.gov).
  • telogen hairs were used from previous cases. DNA from telogen hairs has been like before in Hellmann et al. [A. Hellmann, U. Rohleder, H. Schmitter, M. Wittig, STR typing of human telogen hairs-a new approach, Int J Legal Med 114 (2001) 269-273.]. Briefly, about one cm of a hair fragment containing the telogen root was digested in 500 ⁇ l of TNca buffer. After purification by standard phenol / chloroform method, the DNA was concentrated with a Microcon-30 microconcentrator (Millipore, Eschborn, Germany).
  • STR analysis of artificially degraded DNA was performed using aliquots of DNA from the Pl 18 and HepG 2 cell lines stored by our collaborative European exercise on degraded DNA [PM Schneider, K. Bender, et al. STR analysis of artificially degraded DNA results of a collaborative European exercise, Forensic Int Int 139 (2004) 123-134, K. Bender, MJ. Farfan, PM Schneider, Preparation of degraded human DNA under controlled conditions, Forensic Sei Int 139 (2004) 135-140.].
  • PCR amplification For the amplification of very short tandem repeat systems, the commercially available Quiagen® Multiplex PCR Kit (Quiagen, Hilden, Germany) was used according to the manufacturer's instructions with Applied Bioystems Gene® Amp PCR System 2400/2700 Thermocyclers. The PCR was performed in 25 ⁇ l reaction volume containing 11 ⁇ l of template DNA, 12.5 ⁇ l of Multiplex PCR P.
  • allelic ladders from the commercially available SGM Plus TM (Applied Biosystems) or PowerPlex® 16 (Promega) kits were used as templates. Lead aliquots from the kits were diluted 1 in 1,000,000 and amplified in individual PCR reactions using the same conditions as described for the multiplex PCR, except for a reduced number of 30 cycles. To demonstrate the accuracy of these ladders, the allelic determinations of all loci were compared between samples typified by the SGM Plus TM and the PowerPlex® 16 kit and the new Bioplex-11 multiplex kit. All results were found identical (Figure 1). The allelic ladder for D21S391 was reamplified from a ladder used in a previous study [W. Waiyawuth, L. Zhang, C. Rittner, PM Schneider, Genetic Analysis of the short tandem repeat system D12S391 in the German and three Asian popula- tions, Forensic See Int 94 (1998) 25-31].
  • the allelic ladders were constructed so that they did not overlap.
  • the JOE-labeled systems D8S1179 and D21S11 ranging from 76-120 bases each and from 153-209 bases
  • the 6-FAM labeled systems THO1 and FGA each of 56-95 bases and 124- 262 bases
  • Some STR loci are separated by only a few base pairs, e.g. the 6-FAM-labeled systems D3S1358 and D2S1338 as well as the JOE-labeled systems TPOX and VWA.
  • the fragment sizes of adjacent STR systems are one or two bases outside the tetramer reading frame for allele determination. This was achieved by increasing the sizes of the amplicons of the larger systems by adding up to six bases to the 5 'end of both primers (see Table 2).
  • allelic ladders do not overlap in the background. look at their tetrameric repeat sizes between the 6-plex and the 5-plex reactions when using the same fluorescent dye.
  • the biotinylated products from the PCR reaction were immobilized on streptavidin-coated sepharose beads (Streptavidin Sepharose TM HP 5 Amersham Biosciences Ltd.). Briefly, three microliters of Sepharose beads solution were mixed with 20 ⁇ l of PCR products, binding buffer (10 mM Tris-HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, pH 8.0, 0.1% Tween 20) and water mixed in a final volume of 80 ⁇ l. The mixture was incubated for 15 minutes at room temperature with continuous mixing on a shaker (2000 rpm).
  • the beads were centrifuged off, the supernatant was further purified with the MSB Spin PCRapace Kit (Intritek GmbH, Germany) according to the manufacturer's instructions, and the PCR products were last in 20 ul elution buffer (10 mM Tris-HCl, pH 8,0). Sepharose bead immobilized PCR products were washed twice, first with 150 ⁇ l of wash buffer (10 mM Tris-acetate, pH 7.6) and then with the same volume of 70% ethanol. Finally, the beads were resuspended in 20 ⁇ l elution buffer from the MSB Spin PCRapace Kit.

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Abstract

La présente invention concerne une nouvelle stratégie de détermination de type par STR qui permet à la fois une amplification et une analyse de plusieurs systèmes polymorphes (par ex. onze) présentant des tailles d'amplicon inférieures à 270 pb. Après amplification, la réaction multiplex est divisée en deux ensembles de multiplex STR, puis est analysée de manière séparée. Ce système multiplex a été spécialement développé et testé pour être utilisé dans le cadre de travaux de détermination médico-légaux lorsque l'on ne dispose que de quantités limitées d'ADN ou d'ADN fortement endommagé, par exemple isolé à partir de racines de cheveux en phase télogène.
EP06707244A 2005-02-24 2006-02-24 Procédé pour déterminer le type d'un individu au moyen de loci de microsatellites (str) de l'adn génomique Withdrawn EP1853723A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005008583A DE102005008583B4 (de) 2005-02-24 2005-02-24 Verfahren zur Typisierung eines Individuums mittels short tandem repeat (STR)-Loci der genomischen DNA
PCT/EP2006/001701 WO2006089762A1 (fr) 2005-02-24 2006-02-24 Procede pour determiner le type d'un individu au moyen de loci de microsatellites (str) de l'adn genomique

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