US20130183666A1

US20130183666A1 - Partial genotyping by differential hybridization

Info

Publication number: US20130183666A1
Application number: US13/352,742
Authority: US
Inventors: Marc N. Feiglin; Joseph Mamone; Howard Gamper
Original assignee: Tecan Trading AG
Current assignee: Tecan Trading AG
Priority date: 2012-01-18
Filing date: 2012-01-18
Publication date: 2013-07-18
Also published as: US9783842B2; US20150010908A1; EP2773780B1; EP2773780A1; WO2013107857A1; CN104053790B; CN104053790A

Abstract

In a method of detecting the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, the genomic sample is amplified by PCR and a single stranded target DNA is selected and separated for use in differential hybridization experiments. Subsequent partial genotyping comprises the steps of admixing to the target DNA at least one fluorescent labeled STR probe oligonucleotide and one different fluorescent labeled reference probe oligonucleotide allowing hybridization to the single stranded target DNA in a hybridization experiment. Measurement of the fluorescence intensities of the probes that are bound to the repeat units of the selected STR and normalizing the fluorescence intensity of the STR probes on the base of the reference probe intensity reveals a relative fluorescence signal representing the result of the differential hybridization experiment. Also disclosed are kits for carrying out partial genotyping by differential hybridization.

Description

FIELD OF TECHNOLOGY

The present invention relates to a method of analyzing short tandem repeats according to the preamble of the independent claim or claims. A brief overview over the actual field of technology, commercial kits, DNA separations, recovery of information from degraded DNA, and perspectives of the future dealing with short tandem repeats (STRs) that are sometimes also referred to as micro-satellites or simple sequence repeats (SSRs) is given by John M. Butler in the Mini-Review “Short tandem repeat typing technologies used in human identity testing” (BioTechniques 2007, Suppl. to Vol. 43, No. 4).

RELATED PRIOR ART

The analysis of short tandem repeats (STRs) of individual human genomes is routinely used, e.g. in human identity testing, and in testing of other organisms like plants and cells. Such short tandem repeats are simple sequence motifs of a few up to several dozen repeat units. The human genome comprises thousands of such STRs, which are typically located in non-coding regions. As STRs are polymorph with respect to their number of repeat units, human individuals may be distinguished from each other by the unique number of repeat units per allele and per STR locus. Therefore, the analysis of STRs has turned out to be particularly useful in the identification of human individuals, e.g. in forensic medicine or parentage testing.
Typically, the analysis of STRs involves as a first step the isolation of genomic DNA of human individuals, followed by a Polymerase Chain Reaction (PCR) amplification step. Here, specific, selected STR loci are amplified, and multiplexing (the amplification of multiple STR loci simultaneously) has become routine in biological laboratories. This allows that in a single test a high discrimination rate may be achieved due to the assessment of several STRs in parallel, while only minor amounts of DNA have to be employed. This in turn is of particular relevance in forensic medicine, where often only minor amounts of DNA are available (vast amounts of DNA may be degraded).
Depending on the aim of the analysis (e.g. human identification, parentage testing, population analysis), different STRs may be used. In particular when DNA profiles should be compared among different laboratories, standardization of STR analysis is an important aspect. For example, there are at least seven well established Interpol STR loci that are used for STR analysis in European forensic laboratories (see Gill et al., “The evolution of DNA databases—Recommendations for new European STR loci”, Forensic Science International, 156 (2006), 242-244). This standardization of analyzed STR loci allows a direct comparison of DNA profiles throughout the different laboratories involved.
After the amplification step of selected genomic fragments, the length of each amplified STR is determined. Fragment length determination is widely done using e.g. capillary electrophoresis. Here, the amplified DNA products are separated by electrophoresis and detected by comparison to a standardized allelic ladder. Advantages of DNA length determination using capillary electrophoresis include highly precise sizing (e.g. to less than 1 nucleotide), multiplexing by size by making some amplicons bigger than others but labeling all amplicons with the same fluorescent label (increases throughput). Utilizing capillary electrophoresis provides the advantage that mixtures are much more easily interpreted since intensity and size data are both available to the analyst. However, capillary electrophoresis requires the use of large instruments (e.g. the ABI 3730 Genetic Analyzer, Applied Biosystems). This increases the incurring costs and the complexity of the application. Additionally, the relatively long sample run times reduce the sample throughput and thus can result in backlogs in the respective laboratory. Further advantages of capillary electrophoresis comprise that commonly used instruments with significant installed base in genomic laboratories can be utilized and STR analysis is more easily automatable than prior generation of slab gel electrophoresis. Since capillary electrophoresis does not directly interrogate nucleotide sequences, micro-heterogeneity of the STR due to sequence substitutions are not detected if the STR is of the same length; thus, important information can be lost. Moreover capillary electrophoresis instrumentation is delicate, expensive, and sensitive to dust and movement. The detection window (signal between noise at the low end and maxed out at the high end) is relatively narrow, necessitating expensive, time consuming, and cumbersome quantitative PCR quantification of DNA and normalization to get into the “sweet spot”.
Other approaches include the use of hybridization techniques for STR fragment length determination. For example, in the document WO 96/36731, the number of repeats is determined by hybridizing a target DNA with a unique set of complementary probes containing tandem repeats of known length. If a probe containing more repeats than the target DNA hybridizes, a loop structure is formed, while hybridization of a probe with the identical number of repeats, no loop structure is formed. The length is then identified using the different fluorescent labels of the various probes without using electrophoretic separation. This is a multistep process involving digestion with a nuclease specific to S—S bonds and labeling with a DNA polymerase. This method requires synthesis of a solid supported oligonucleotide array, and therefore cannot be done in solution.
In the document U.S. Pat. No. 6,395,493 B1 a method for determination of length polymorphism in DNA is disclosed, which also involves a hybridization reaction. This document describes an assay that involves the use of a silicon microchip composed of an arrayed set of electrodes that each contain a unique “capture probe” for each possible allele of each possible STR loci of interest. For example, in order to determine which of the possible eight alleles at the TPOX locus (e.g., 6-13 repeats) are present; eight different probe sites are required. The DNA sample of interest is amplified and then washed over the chip. It will hybridize to the electrodes with complementary capture probes. The “capture probe” captures the PCR-amplified STR allele by binding to the repeat region and 30-40 bases of the flanking region. After hybridization, an “electronic stringency” is then applied to each probe site by simply adjusting the electric field strength. Samples that are not a perfect match for the probe will be denatured and driven away from the probe.
After removing unbound and denatured DNA, a mixture of “reporter probes” is washed over the chips. The “reporter probe” contains 1-3 repeat units, some flanking sequence and a fluorescent dye. This probe will hybridize to the STR allele of DNA captured on the chip and generates a fluorescence signal at the probe site that can be interpreted to yield the sample's genotype. The read-out provides a genotype that corresponds to the number of repeats present in the sample even though no size-based separation has been performed.
In this method, an array of capture probes must be “printed” on the surface of the reaction vessel and the DNA is subsequently washed over this array. The Tecan method binds the DNA to any surface and then washes the probes over the DNA. In consequence, this Nanogen method requires pre-printing or purchase of a special pre-printed array. With this Nanogen assay, the intensity of the read-out signal is limited by the number of capture oligonucleotides printed on each electrode. Further amplification of the DNA sample cannot increase signal beyond the number of capture electrodes. This Nanogen method requires special instrumentation to denature mismatched hybrids prior to washing. The Tecan method requires no such special instrumentation. The Nanogen method can be performed in a single reaction vessel (i.e. microarray slide).

OBJECTS AND SUMMARY OF THE PRESENT INVENTION

It is an object of the present invention to suggest a method of determining the number of tandem repeats in a nucleic acid probe.
According to a first aspect, this object is achieved by a method of detecting the number of repeat units in a selected short tandem repeat (STR) in a genomic sample according to the present invention. The method as herein disclosed comprises the steps of:

a) providing at least one:
- a1) genomic sample containing the selected STR;
- a2) set of polymerase chain reaction- (PCR) oligonucleotides for carrying out PCR amplification of the selected genomic sample with the short STR sequence;
- a3) STR probe oligonucleotide (P1,P1′) with individual fluorescent label;
- a4) reference probe oligonucleotide (P2) with a different fluorescent label;
b) amplifying the genomic sample with the selected STR sequence provided in step a1) by PCR using the set of PCR oligonucleotides provided in step a2), denaturing the amplified double stranded sample DNA for generating single stranded DNA, selecting and separating a single stranded target DNA for use in hybridization experiments;
c) carrying out partial genotyping by differential hybridization that comprises the steps:
- c1) mixing an amount of the single stranded target DNA selected and separated in step b) with the STR probe oligonucleotides provided in step a3) and the reference probe oligonucleotide (P2), and allowing hybridization to the single stranded target DNA in a hybridization experiment;
- c2) measuring the intensity of the fluorescence provided by the labeled STR probe oligonucleotides that are bound to the repeat units of the selected STR;
- c3) measuring the intensity of the different fluorescence provided by the labeled reference probe oligonucleotide (P2) that is bound to a flanking sequence of the target DNA; and
- c4) normalizing the intensity measured of the STR probe fluorescence in step c2) based on the intensity measured of the different fluorescence in step c3) and revealing a relative fluorescence signal representing the result of the differential hybridization experiment.

According to a second aspect, this object is achieved by proposing kits for carrying out partial genotyping by differential hybridization as herein disclosed.
Additional features of the present invention and preferred embodiments are herein disclosed as well.
Advantages of the method according to the present invention comprise:

- The assessment of the number of repeats may be done using standard microplate fluorescence readers instead of large capillary electrophoresis devices. Such fluorescence readers are often already available in typical forensic, medical or diagnostic laboratories, and of even more importance: such a standard microplate fluorescence reader costs about 10% of a capillary electrophoresis device (the latter being about US $ 200,000).
- The analysis time can be reduced, as first results may be obtained faster than in capillary electrophoresis.
- The method of the current invention is simpler than conventional analysis methods, as the method is not enzymatic. This method is sequence specific (versus sizing), so microvariants can be detected by inclusion of additional probes.
- The present method is less sensitive to input DNA and provides a broader dynamic range). Because of this restriction on capillary electrophoresis, an additional step of quantifying the DNA prior to capillary electrophoresis is required; this is not necessary when applying the method of the current invention.
- The presented method is faster (not including the time required by both methods for sample preparation, e.g. purification and amplification), a user can analyze the 13 CODIS loci on 7 different samples on a single 384 well microplate or 29 samples on a 1536 well plate. Such plates can be measured on a typical microplate reader in less than a minute or two. High throughput capillary electrophoresis devices can process up to 16 samples per run with each run taking 1-2 hours.
- In contrast to known multistep methods involving digestion with a nuclease that is specific to S—S bonds and labeling with a DNA polymerase, the method of the current invention is much simpler and accomplished with one hybridization experiment.
- Known multistep methods may require synthesis of a solid supported oligonucleotide array or “printing” such a capture probe array on the surface of the reaction vessel, the DNA then is subsequently washed over this array. In contrast, the present invention binds the DNA to any surface and then washes the probes over the DNA; the hybridization is done in solution and pre-printing or purchasing of a special pre-printed array is not necessary.
- Known multistep methods require special instrumentation to denature mismatched hybrids prior to washing. The inventive method does not require such special instrumentation. In fact, the presented method could be designed as a homogenous assay using quenching probe chemistries such as available from Molecular Beacons or Foerster Resonance Energy Transfer (FRET).

BRIEF INTRODUCTION OF THE ATTACHED DRAWINGS

With the help of the attached drawings, the preferred embodiments of the method and kits of the present invention are illustrated without narrowing the scope of the present invention. It is shown in:

FIG. 1 a schematic overview over typical kits of oligonucleotides for carrying out the method of partial genotyping of the human CSF1PO STR by differential hybridization;

FIGS. 2-11 a schematic illustration of some possible hybridization results that are theoretically achievable by the method and kits of the present invention, wherein it is shown in:

FIG. 2 a group A CSF1PO target DNA with 2 tetranucleotide repeats without blocking and flanking oligonucleotides (or oligos for short), and with no hybridization;

FIG. 3 a group B CSF1PO target DNA with 4 tetranucleotide repeats with one blocking oligonucleotide (or oligo for short), and with no hybridization;

FIG. 4 a group C CSF1PO target DNA with 7 tetranucleotide repeats with one blocking oligonucleotide (or oligo for short), and with hybridization of one STR probe;

FIG. 5 a group G CSF1PO target DNA with 12 tetranucleotide repeats with one blocking oligo, and with hybridization of two STR probes;

FIG. 6 a group J CSF1PO target DNA with 16 tetranucleotide repeats with one blocking oligo, and with hybridization of three STR probes;

FIG. 7 a group J CSF1PO target DNA with 16 tetranucleotide repeats with two flanking oligos, and with hybridization of four STR probes;

FIG. 8 a group J CSF1PO target DNA with 16 tetranucleotide repeats with two blocking oligos, and with hybridization of two STR probes;

FIG. 9 a group J CSF1PO target DNA with 16 tetranucleotide repeats with one flanking oligo and one blocking oligo, and with hybridization of three STR probes; and in

FIG. 10 a group J CSF1PO target DNA with 16 tetranucleotide repeats with one blocking oligo and one flanking oligo, and with hybridization of three STR probes;

FIG. 11 a target DNA with a non STR island sequence located in the STR fragment of a group J CSF1PO target DNA with 16 tetranucleotide repeats; with one blocking oligo and one flanking oligo, with hybridization of three STR probes, and with projected hybridization of an insert probe with a nucleotide sequence complimentary to the island sequence;

FIG. 12 diagram showing a combination of theoretical expectations and achieved results of experiments as carried out with FAM-labeled 16-mer probes (5′-AGAT)₄hybridized to a series of chemically synthesized single-stranded DNAs which contained 2-16 ATCT repeats, wherein it is shown in:

FIG. 12A the groups A and B of target DNA;

FIG. 12B the groups C, D, E, and F of target DNA; and in

FIG. 12C the groups G, H, I and J of target DNA;

FIG. 13 a table diagram displaying the results obtained from allele pairs in STR assays.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to the detection of the number of repeats in selected STR loci. According to the present invention, the number of repeat units is correlated to the signal intensity of parallel hybridization experiments. By comparison to a normalization probe, exact number of repeats can be easily determined.
Selection of STR Loci:
For human identification it is proposed to use the STR loci that are generally accepted by the respective law enforcement agency. The two major sets are the 13 FBI (US Federal Bureau of Investigation) CODIS Loci and the 10 FSS (United Kingdom Forensic Science Service) SGM and SGM plus loci. Non-human DNA testing and microbial forensics is described by John M. Butler in “Forensic DNA Typing, Biology, Technology, and Genetics of STR Markers” (Elsevier Academic Press, Second Edition 2005; see chapter 11, pages 299-330). There, cat and dog STRs are described (and the sources referenced) as well as plant STRs (e.g. Cannabis sativa) and it is pointed out that “as with human STRs, marijuana STR markers are highly polymorphic, specific to unique sites in the genome, and capable of deciphering mixtures. A heaxanucleotide repeat marker showed repeat units ranging from 3-40 in 108 tested marijuana samples, and primers amplifying this locus produced no cross-reactive amplicons from other 20 species of plants tested (Hsieh et al 2003)”. From microbial forensics, first steps are reported in connection with bioterrorism, including genome sequencing of Bacillus anthracis (anthrax) and phylogenetic analyses of viral strains of HIV.
Among the various types of STR systems, tetranucleotide repeats (4 repeat units in the core repeat) have become more popular than di- or trinucleotides (2 or 3 repeat units). Penta- and hexanucleotides (5 or 6 repeat units) repeats are less common in the human genome but are being examined by some laboratories (see Butler 2005, page 89, 3^rdparagraph).
PCR:
A PCR amplification step is necessary when working with STR systems because genomic DNA would be too complex for hybridization assays. Multiplex PCR, where a defined number or a combination of STR loci are treated simultaneously is possible. There actually is no maximum or minimum number of STRs; everything what is empirically possible is preferred. A large number of STR markers have been characterized by academic and commercial laboratories for use in disease and gene location studies. For example, the Marshfield Medical Research Foundation in Marshfield, Wis. (http://research.marshfieldclinic.org/genetics) has gathered geno-type data ob over 8000 STRs that are scattered across the 23 pairs of human chromosomes (see Butler 2005, page 86). There exist many commercial kits, e.g. from Applied Biosystems, Promega, and Qiagen, to accomplish the appropriate multiplex.
Used Oligonucleotides:
In a given experiment, one of the repeat probes is used with the normalization probe, and one or more of the blockers OR one or more of the flankers. When different oligonucleotides are used as a mixture and given together to each experimental sample according to the present invention, the preferred oligonucleotides are chosen such that the melting temperature (T_m) is sufficiently high to bind in all cases. Accordingly, the temperature of the experiment should be lower than T_m(detailed description see below).
Strategy of Partial Genotyping of the Human CSF1PO STR by Differential Hybridization:
CSF1PO is a short tandem repeat (STR) composed of 5-16 consecutive runs of the 5′-ATCT-3′-TAGA tetramer located at a unique position in human chromosome 5.
To partially differentiate between the repeat numbers, a novel hybridization approach was applied. In this approach, a FAM-labeled 16-mer probe (5′-AGAT)₄was hybridized to a series of chemically synthesized single-stranded DNAs which contained 2-16 ATCT repeats. Each of these 5′-ATCT-3′ repeats was embedded in the middle of a longer unrelated sequence which was biotinylated at the 5′-end (see FIG. 1). In FIG. 1 for illustration purposes, tetranucleotide repeats of the STRs in the single-stranded target DNA are indicated in each case as one box and sixteen such tetranucleotide 5′-ATCT-3′ repeats in the single-stranded target DNA are indicated by the boxes numbered 1-16. The site at the 5′-end of the single-stranded target DNA is marked with B* that stays for biotinylated. As well know in the art of nucleic acid manipulation in biochemistry, such biotinylation enables binding of the single-stranded target DNA molecules to streptavidine-coated magnetic beads. The DNA fragment from the 5′-end to the beginning of the STRs is called 5′-flanking region and the DNA fragment from the 3′-end of the STRs to the 3′-end of the DNA is called 3′-flanking region.
In FIG. 1 it is schematically shown an overview over typical kits of oligonucleotides for carrying out the method of partial genotyping of the human CSF1PO STR by differential hybridization. Such a kit at least comprises an STR probe P1 for hybridizing with a target STR and a reference probe P2 for identification of the relevant single-stranded target DNAs. It is preferred that the STR probe P1 contains the four nucleotides 5′-AGAT-3′ and that the reference probe P2 is labeled with FAM at the 3′-end. FAM (Applied Biosystems Inc.) is a fluorescein derivative and a member of the xanthene fluorescent dyes. Preferably, the reference probe P2 is configured as a 25-mer which is complementary to the region of the target DNA indicated in FIG. 1, e.g. the 3′-flanking region. It is further preferred that the reference probe P2 is labeled with a cyanine dye, e.g. Cy5.
In general, any discernible labeling that allows discrimination of the two probes can be applied to the STR probe P1 and to the reference probe P2. These can be fluorescent dyes as already indicated; however, also donor-acceptor fluorescent pairs e.g. FAM-3-TAM, or FAM-3-ROX, or FAM-4-ROX as disclosed in U.S. Pat. No. 5,654,419 can be used (the rhodamine derivatives TAM and ROX are dyes of Applied Biosystems Inc.). Even if some alternatives to fluorescence labeling may exist, fluorescence is preferred because of its ability of providing multiple colors, being fast, and being sensitive. However, any sort of measurable label that can be attached to primers and that can be multiplexed could be used, i.e. radioactive, luminescent, chromogenic, tagged beads, etc.
Preferably, the kit also comprises at least one blocking oligonucleotide B1,B2 for hybridizing with a fragment of a target STR and with a fragment of the relevant single-stranded target DNA. Preferably and as depicted in FIG. 1, the blocking oligonucleotide B1 bridges the 5′-end of the STRs with the 5′ flanking region of the DNA and the blocking oligonucleotide B2 bridges the 3′-end of the STRs with the 3′-flanking region of the DNA. Of special preference are blocking oligonucleotides B1,B2 that are 25-mers which are complementary to the regions of the target DNA indicated in FIG. 1.
Preferably, the kit also comprises at least one flanking oligonucleotide F1,F2 for hybridizing with a fragment of the relevant single-stranded target DNA adjacent to the target STRs. Preferably and as depicted in FIG. 1, the flanking oligonucleotide F1 hybridizes to the 5′-flanking region from the 5′-end of the STRs in direction to the 5′-end of the single-stranded target DNA and the flanking oligonucleotide F2 hybridizes to the 3′-flanking region from the 3′-end of the STRs in direction to the 3′-end of the single-stranded target DNA. Of special preference are flanking oligonucleotides F1,F2 that are 25-mers which are complementary to the regions of the target DNA indicated in FIG. 1.
Importantly, the flanking oligonucleotides F1,F2 hybridize immediately adjacent to the CSF1PO repeat while each of the blocking oligonucleotides B1,B2 hybridizes to 12 nucleotides (3 repeats) of the CSF1PO sequence and to 13 nucleotides of the 5′- or 3′-flanking target sequences. It is also important to note that on the same 5′- or 3′-flanking region of the target DNA, only a blocking oligonucleotide B1,B2 or a flanking oligonucleotide F1,F2 can hybridize; thus, either two blocking oligonucleotides, i.e. B1 & B2, two flanking oligonucleotides, i.e. F1 & F2, or one blocking oligonucleotide plus one flanking oligonucleotide, i.e. B1+F2 or F1+B2 are to be used.
The following kits are preferred:

a) STR probe P1 and reference probe P2 (also referenced as “XX”);
b1) STR probe P1, reference probe P2, with blocking oligonucleotide B1 (also referenced as “BX”);
b2) STR probe P1, reference probe P2, with blocking oligonucleotide B2 (also referenced as “XB”);
b3) STR probe P1, reference probe P2, with blocking oligonucleotides B1 & B2 (also referenced as “BB”);
c1) STR probe P1, reference probe P2, with flanking oligonucleotide F1 (also referenced as “FX”);
c2) STR probe P1, reference probe P2, with flanking oligonucleotide F2 (also referenced as “XF”);
c3) STR probe P1, reference probe P2, with flanking oligonucleotides F1 & F2 (also referenced as “FF”);
d) STR probe P1, reference probe P2, with blocking oligonucleotide B1, and flanking oligonucleotide F2 (also referenced as “BF”);
e) STR probe P1, reference probe P2, with blocking oligonucleotide B2, and flanking oligonucleotide F1 (also referenced as “FB”).

Depending on the actual number of tetranucleotide repeats present in the target DNA, the extent of hybridization of the 16-mer CSF1PO specific STR probe P1 in the presence of the various combinations of blocking oligonucleotides B1,B2 and flanking oligonucleotides F1,F2, the target DNAs theoretically can be divided into 10 different groups A-3 thus partially genotyping this tetranucleotide repeat (see Table 1).

TABLE 1

Identification of CSF1PO repeat number with a 16-mer probe

CSF1PO

Repeat

No Blocker

One Blocker

Two Blockers

Target	Number	Bases	Hybrids	Bases	Hybrids	Bases	Hybrids	Group

12	2	8	0	0	0	0	0	A
13	3	12	0	0	0	0	0
14	4	16	1	4	0	0	0	B
1	5	20	1	8	0	0	0
15	6	24	1	12	0	0	0
16	7	28	1	16	1	4	0	C
2	8	32	2	20	1	8	0	D
17	9	36	2	24	1	12	0
18	10	40	2	28	1	16	1	E
19	11	44	2	32	2	20	1	F
3	12	48	3	36	2	24	1	G
20	13	52	3	40	2	28	1
21	14	56	3	44	2	32	2	H
22	15	60	3	48	3	36	2	I
4	16	64	4	52	3	40	2	J

In Table 1, one repeat corresponds to four base pairs (bp); a 16-mer probe P1 hybridizes to 4 repeats; each blocking oligonucleotide B1,B2 hybridizes to 3 repeats (see also FIG. 1).
The attached FIGS. 2 to 10 schematically illustrate some possible hybridization results that are theoretically achievable by the method and kits of the present invention and that are listed in Table 1.
According to FIG. 2, a group A CSF1PO target DNA with 2 tetranucleotide repeats incubated with kit a) (only containing STR probe P1 and reference probe P2) is expected to show no hybridization, because there are not enough STRs present in the target DNA for achieving substantial hybridization. Addition of blocking oligos or flanking oligos would change the result.
According to FIG. 3, a group B CSF1PO target DNA with 4 tetranucleotide repeats incubated with kit b1) (containing STR probe P1, reference probe P2, with one blocking oligonucleotide B1) is expected to show no hybridization, because there are not enough STRs present in the target DNA for achieving substantial hybridization. The addition of the other blocking oligo B2 (i.e. using kit b3) or the addition of the flanking oligo F2 (i.e. using kit d) would not change the result.
According to FIG. 4, a group C CSF1PO target DNA with 7 tetranucleotide repeats incubated with kit b2) (containing STR probe P1, reference probe P2, with one blocking oligonucleotide B2) is expected to show a single hybridization per target DNA strand, because there are just enough STRs present in the target DNA for achieving one substantial hybridization. The addition of the flanking oligo F1 (i.e. using kit e) would not change the result. In contrast, the addition of the other blocking oligo B1 (i.e. using kit b3) would considerably change the result, because then there would not be enough STRs present in the target DNA any more for achieving substantial hybridization.
According to FIG. 5, a group G CSF1PO target DNA with 12 tetranucleotide repeats incubated with kit b1) (containing STR probe P1, reference probe P2, with one blocking oligonucleotide B1) is expected to show two hybridizations per target DNA strand, because there are enough STRs present in the target DNA for achieving two substantial hybridizations. The addition of the flanking oligo F2 (i.e. using kit d) would not change the result. In contrast, the addition of the other blocking oligo B1 (i.e. using kit b3) would considerably change the result, because then there would be only enough STRs present in the target DNA any more for achieving one substantial hybridization.
According to FIG. 6, a group J CSF1PO target DNA with 16 tetranucleotide repeats incubated with kit b1) (containing STR probe P1, reference probe P2, with one blocking oligonucleotide B1) is expected to show three hybridizations per target DNA strand, because there are enough STRs present in the target DNA for achieving three substantial hybridizations. The addition of the flanking oligo F2 (i.e. using kit d) would not change the result. In contrast, the addition of the other blocking oligo B1 (i.e. using kit b3) would considerably change the result, because then there would be only enough STRs present in the target DNA any more for achieving two substantial hybridizations.
According to FIG. 7, a group J CSF1PO target DNA with 16 tetranucleotide repeats incubated with kit c3) (containing STR probe P1, reference probe P2, with two flanking oligonucleotides F1 and F2) is expected to show four hybridizations per target DNA strand, because there are just enough STRs present in the target DNA for achieving four substantial hybridizations. Addition of blocking oligos B1 and/or B2 is not allowed because of competition with the flanking oligonucleotides F1 and/or F2 during hybridization.
According to FIG. 8, a group J CSF1PO target DNA with 16 tetranucleotide repeats incubated with kit b3) (containing STR probe P1, reference probe P2, with two blocking oligonucleotides B1 and B2) is expected to show two hybridizations per target DNA strand, because there just are enough STRs present in the target DNA for achieving two substantial hybridizations. Addition of flanking oligos F1 and/or F2 is not allowed because of competition with the blocking oligonucleotides B1 and/or B2 during hybridization.
According to FIG. 9, a group J CSF1PO target DNA with 16 tetranucleotide repeats incubated with kit e) (containing STR probe P1, reference probe P2, with one flanking oligonucleotide F1 and one blocking oligonucleotide B2) is expected to show three hybridizations per target DNA strand, because there are just enough STRs present in the target DNA for achieving three substantial hybridizations. Addition of a blocking oligo B1 and/or of a flanking oligo F2 is not allowed because of competition with the flanking oligonucleotide F1 and/or with the blocking oligonucleotide B2 during hybridization.
According to FIG. 10, a group J CSF1PO target DNA with 16 tetranucleotide repeats incubated with kit d) (containing STR probe P1, reference probe P2, with one blocking oligonucleotide B1 and one flanking oligonucleotide F2) is expected to show three hybridizations per target DNA strand, because there are just enough STRs present in the target DNA for achieving three substantial hybridizations. Addition of a blocking oligo B2 and/or of a flanking oligo F1 is not allowed because of competition with the flanking oligonucleotide F2 and/or with the blocking oligonucleotide B1 during hybridization.
Having practiced enough with theoretical expectations, inspection of the results of some practical experiments shall now be made. The diagrams of FIG. 12 show a combination of the above exercised theoretical expectations and of the actually achieved results. These results have been achieved from experiments that were carried out with FAM-labeled 16-mer probes (5′-AGAT)₄hybridized to a series of chemically synthesized single-stranded DNAs which contained 2-16 ATCT repeats.

Experiments:

Initially, a series of single-stranded DNAs which contained 2-16 ATCT repeats was chemically synthesized. Each of these 5′-ATCT-3′ repeats was embedded in the middle of a longer unrelated sequence which was biotinylated at the 5′-end (see FIG. 1 with a target DNA having 16 tetramer ATCT-STRs).
Prior to hybridization, each of the single-stranded target DNAs as depicted in FIG. 1 was loaded onto a suspension of washed, streptavidine-coated magnetic beads. The binding reactions utilized 0.5 mg of beads and 80 pMoles of each DNA target. It should be noted that capture efficiency usually is greater than 90%.
In addition to a 16-mer STR probe P1 (as indicated in FIG. 1 as well), each hybridization reaction included a longer Cy5-labeled reference probe P2 and either two flanking oligonucleotides (or oligomers or oligos for short) (F1 & F2), two blocking oligos (B1 & B2), or one flanking plus one blocking oligomer (F1+B2 or B1+F2). Accordingly, the kits b3), c3), d), or e) as defined above have been utilized for hybridization with the single-stranded target DNAs. It is noted here, that alternatively, the kit b1) could have been utilized instead of kit d) and the kit b2) could be utilized instead of kit e), because the same results would have been expected.
The reference oligomers P2, flanking oligomers F1,F2, and blocking oligomers B1,B2 were 25-mers complementary to the regions of the target DNA indicated in FIG. 1. The flanking oligomers F1,F2 hybridized immediately adjacent to the CSF1PO repeat while each of the blocking oligomers B1,B2 hybridized to 12 nucleotides (3 repeats) of the CSF1PO sequence and to 13 nucleotides of the adjacent flanking target sequence.
For hybridization, 160 pMoles of the Cy5-labeled reference probe P2 together with 160 pMoles each of the desired combination of blocking oligonucleotides B1,B2 and flanking oligonucleotides F1,F2 were added to 0.5 mg of streptavidine-coated magnetic beads previously loaded with 80 pMoles of a specific single-stranded target DNA. Hybridization was conducted for 5 min at 65° C. followed by 15 min at 37° C. in 100 μl of Buffer A (10 mM Hepes pH 8.0, 50 mM NaCl, 10 mM MgCl₂). Next, 160 pMoles of FAM-labeled STR probe P1 was added to the bead suspension. Hybridization of this STR probe P1 to the target DNA was conducted for 15 min at 47° C. After removal of the hybridization solution, the beads were incubated for 15 min at 47° C. in 100 μl of fresh Buffer A. Finally, bound probes were eluted from the washed beads by incubation for 10 min at 65° C. in Buffer B (10 mM Hepes pH 8.0, mM NaCl). Supernatants containing eluted probes were collected and transferred to a microtiter plate for reading of FAM and Cy5 fluorescence in a TECAN INFINITE® 200 microplate reader (Tecan Austria GmbH, Groedig, Austria).
On the horizontal axis of the diagrams in FIG. 12, the number of STR repeats per target DNA strand is indicated in each case (i.e. 2-16) and assigned to the groups as defined in Table 1 (i.e. A-J). For each group of target DNA strands with a particular number of STR repeats, the results achieved with the utilized kits c3), e), d), or b3) are indicated as vertical-bar graphs (in this order). Alternatively, the four vertical-bar graphs can be named FF, FB, BF, or BB (in this order) according to the utilized flanking oligos “F” and/or blocking oligos B. Above each vertical-bar graph, the expected theoretical result is indicated in numbers of hybridizations per target DNA strand. On the vertical axis of the diagrams in FIG. 12, the relative fluorescence signal (the quotient of the measured intensities FAM/Cy5) is indicated for each one of the 60 Experiments.
FIG. 12A shows the target DNAs of the groups A (2-3 STR repeats) and B (4-6 STR repeats) of target DNA as referred to in the Table 1. A perfect match between the tetramer STR probe P1 and the target DNAs would result, if for hybridization the number of STRs in the target DNA is at least 4. It was thus expected that the target DNAs of the group A will show no hybridization and that the target DNAs of the group B will only show hybridization for kit c3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of kit c3) in the group B is strikingly higher than the height of the vertical-bar graphs representing the use of one of the kits e), d), or b3). Nevertheless, there is some noticeable hybridization in group A; however if compared with the results of group B, very little signal is achieved indeed. In consequence, the results expected for the groups A and B are regarded as clearly verified.
FIG. 12B shows the target DNAs of the groups C (7 STR repeats), D (8 or 9 STR repeats), E (10 STR repeats), and F (11 STR repeats) of target DNA as referred to in the Table 1. A perfect match between the tetramer STR probe P1 and the target DNAs would result, if for hybridization the number of STRs in the target DNA is at least 4 (one hybridization per target DNA strand) or 8 (two hybridizations per target DNA strand).
It was thus expected that the target DNAs of the group C will show one hybridization when utilizing the kits c3), e), or d) and no hybridization when utilizing the kit b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits c3), e), or d) in the group C is strikingly higher than the height of the vertical-bar graph representing the use of the kit b3). It was also expected that the target DNAs of the group D will show two hybridizations when utilizing the kit c3), one hybridization when utilizing the kits e) or d), and no hybridization when using the kit b3). The height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit c3) in the group D is about double the height of the vertical-bar graphs representing the use of the kits e) or d); the height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit b3) is considerably lower than the height of the vertical-bar graphs representing the use of the kits e) or d). Even if there is some noticeable hybridization in group D when utilizing the kit b3), if compared with the results of the other kits very little signal is achieved, however. In consequence, the results expected for the groups C and D are regarded as clearly verified.
It was also expected that the target DNAs of the group E will show two hybridizations when utilizing the kit c3) and one hybridization when utilizing the kits e), d), or b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits c3) in the group E is about double the height of the vertical-bar graphs representing the use of the kits e), d) or b3). In consequence, the results expected for the group E are regarded as clearly verified.
It was further expected that the target DNAs of the group F will show two hybridizations when utilizing the kits c3), e), or d) and one hybridization when utilizing the kit b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits c3), e), or d) in the group F is about double the height of the vertical-bar graph representing the use of the kit b3). In consequence, the results expected for the group F are regarded as clearly verified.
FIG. 12C shows the groups G (12-13 STR repeats), H (14 STR repeats), I (15 STR repeats), and J (16 STR repeats) of target DNA as referred to in the Table 1. A perfect match between the tetramer STR probe P1 and the target DNAs would result, if for hybridization the number of STRs in the target DNA is at least 4 (one hybridization per target DNA strand), 8 (two hybridizations per target DNA strand), 12 (three hybridizations per target DNA strand), or 16 (four hybridizations per target DNA strand).
It was expected that the target DNAs of the group G will show three hybridizations when utilizing the kit c3), two hybridizations when utilizing the kits e) or d), and one single hybridization when utilizing the kit b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits e) or d) in the group G in each case is about equal and double the height of the vertical-bar graphs representing the use of the kit b3). The height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit c3) in the group G in each case is considerably higher than the height of the vertical-bar graphs representing the use of the kits e) or d) and about triple the height of the vertical-bar graphs representing the use of the kit b3). In consequence, the results expected for the group G are regarded as verified.
It was expected that the target DNAs of the group H will show three hybridizations when utilizing the kit c3) and two hybridizations when utilizing the kits e), d), or b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits e), d), or b3) in the group H is about equal. The height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit c3) in the group H is considerably higher than the height of the vertical-bar graphs representing the use of the kits e), d) or b3). In consequence, the results expected for the group H are regarded as verified.
It was expected that the target DNAs of the group I will show there hybridizations when utilizing the kit c3), e), or d) and two hybridizations when utilizing the kit b3). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits c3), e), or d) in the group I is about equal. The height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit b3) in the group I is considerably lower than the height of the vertical-bar graphs representing the use of the kits c3, e), or d). In consequence, the results expected for the group I are regarded as verified.
It was expected that the target DNAs of the group J will show four hybridizations when utilizing the kit c3) (compare with FIG. 7), three hybridizations when utilizing the kit e), or d) (compare with FIG. 9 or 10) and two hybridizations when utilizing the kit b3) (compare with FIG. 8). The height of the vertical-bar graphs (relative fluorescence signals) representing the use of the kits e) or d) in the group J is about equal and considerably higher than the height of the vertical-bar graph representing the use of the kit b3). The height of the vertical-bar graph (relative fluorescence signal) representing the use of the kit c3) in the group J is considerably higher than the height of the vertical-bar graphs representing the use of the kits e) or d). In consequence, the results expected for the group J are regarded as verified.
The above analysis has been discussed on the base of the FIG. 12, which summarizes the relative ratios of FAM to Cy5 fluorescence obtained from each hybridization reaction. Since four ratios have been acquired for each target DNA, the target DNAs can be divided into the 10 groups A-3 thus providing an indication of the genotype. The reduction in hybridization efficiency of the STR probe P1 with increased repeat numbers (see particularly FIG. 12C) might be overcome by increasing the ratio of STR probes P1 to DNA targets. The hybridization efficiency of the 16-mer STR probe P1 is estimated to be probably less that 25%.
The preferred STR probe oligonucleotide P1 as utilized:

- is at least one oligo, which consists of a sequence complementary to the sequence of the repeat unit of the selected STR;
- has a complementary sequence that consists of a specific number n of repeat units and, if the STR is a tetramer repeat, the preferred specific number n is at least 4, because n=3 (12 nucleotides in length) would result in a very low T_m;
- the preferred number of repeat units in an STR probe P1 is 4; however, there could be more or less repeat units if the resulting oligonucleotide is long enough to bind at experiment temperatures;
- has a number of repeat units that depends from the type of selected STR, because not all STR's are simple repeats;
- is labeled with a marker of a first fluorescence (e.g. FAM).

The preferred reference oligonucleotide P2 as utilized:

- is labeled with a marker of a second fluorescence (e.g. Cy5);
- serves for normalization of the used amount of DNA in each experiment;
- comprises (preferably consists of) a sequence complementary to the 5′- or 3′-flanking sequence of the target DNA, but without sequences of STRs or of the flanking oligonucleotides;
- could be contiguous with the flanking oligonucleotide; however some distance to the flanking oligonucleotide is preferred;
- must not reach into the sequence of the PCR primer that is used to generate the amplicon;
- does not have a minimum or maximum length; the melting point T_mshould be sufficient that binding is stoichiometric at experimental temperature;
- is used in every experiment.

The preferred blocking oligonucleotide B1,B2 as utilized:

- is for use in addition to the STR probe oligonucleotide P1, e.g. in a parallel control experiment;
- comprises a sequence complementary to the sequence of the repeat unit of the selected STR, the complementary sequence consists of a specific number of repeat units, preferably n=3; however, there could be any number of repeats;
- in all cases also includes some sequence complementary to one of the flanking regions of the target DNA;
- when used in a control experiment together with the STR probe oligo P1, enters a competitive, stronger binding.

The preferred flanking oligonucleotides F1,F2 as utilized:

- reduce any intramolecular secondary structure of the single stranded target DNA from which the antisense strand was stripped away and removed;
- as a set of 3′- and 5′-flanking oligonucleotides F1,F2 was utilized for multiple DNA alleles that are different in numbers of repeats; the set of flanking oligonucleotides always was the same;
- prevent single stranded DNA from forming secondary structures around a particular STR region;
- preferably have a length (e.g. 25 nucleotides) that is designed to hybridize at assay temperature (specific T_mnot being a critical factor);
- if being a 5′-flanking oligo, comprises (preferably consists of) a sequence complementary to the 5′ flanking sequence of a selected STR of the single DNA strand;
- if being a 3′-flanking oligo comprises (preferably consists of) a sequence complementary to the 3′ flanking sequence of the selected STR of the single DNA strand;
- do not comprise sequences that are complementary to the repeat unit sequence;
- do not comprise a fluorescent label.

When carrying out the method of the present invention, the number of repeat unit sequences of the target DNA available for the STR probe oligo P1 is reduced by the addition of blocking oligos B1,B2. When the number of repeat units in the blocking oligo is known, the number of repeat units which are not available any more after binding of the blocking oligo is known too. In any case for the 3′-end and for the 5′-end of the STR fragment of the target DNA, one blocking oligo B1,B2 or one flanking oligo F1,F2 may be used. Thus, the reduction of available repeat units on the target DNA results in a reduction of fluorescence intensity compared to an experiment carried out only with the STR probe oligo but without blocking oligo, and the measured difference of fluorescence intensity is used to determine the number of repeats in the STR (comparison of max. intensity_{probe oligo alone}vs. reduced intensity_{probe oligo+blocking oligo}).
Insert Probes P3:
An insert probe P3 (see FIG. 11) is a specific oligonucleotide probe for “islands” of non-repeat nucleotide sequence within the structure of the STR repeat region (in the following it is referred to these as “more complex” STR's) of a single strand target DNA.
The insert probe P3:

- has a specific sequence that is designed to bind specifically at the “island” within the STR repeat region at the selected assay temperature;
- definitely contains the base(s) complimentary to the island;
- is labeled with a fluorescent label (e.g. Cy3);
- can have two flanking complements to STR repeats (e.g. two repeat units on both ends of the insert probe P3) or possibly even only a portion of a complement to an STR repeat (on one or both ends of the insert probe P3;
- is used for those loci where an “island” might be found;
- has a known insert sequence; the specific sequences of possible alleles have been published and John Butler at NIST maintains a database of such core loci at http://www.cstl.nist.gov/strbase (one allele being CSF1PO). The FBI has published thirteen core loci for the Combined DNA Index System (CODIS) database. STR Fact Sheets for all thirteen loci are available on-line (For more information, see: Butler, J. M. (2006) Genetics and genomics of core STR loci used in human identity testing. J. Forensic Sci. 51(2): 253-265).

In general, the oligos (blockers and probes) are designed so that each possible STR can be uniquely identified with a minimum number of probes. For carrying out the above discussed experiments, a number of oligonucleotides have been chosen for model the CSF1PO test system (5′-AGAT-375′-ATCT-3′ repeat flanked by artificial sequences). These oligonucleotides are described by the sequence listing attached to this patent application. This sequence listing comprises:
SEQ ID: NO 1, a reference target strand with 5 AGAT repeats (not synthesized);
SEQ ID: NO 2, a 16-mer probe to ATCT repeat (STR probe P1);
SEQ ID: NO 3, a 20-mer probe to ATCT repeat;
SEQ ID: NO 4, a 25-mer 5′-complementary oligo (flanking oligo F1);
SEQ ID: NO 5, a 25-mer 5′-blocking oligo (blocking oligo B1);
SEQ ID: NO 6, a 25-mer 3′-complementary oligo (flanking oligo F2);
SEQ ID: NO 7, a 25-mer 3′-blocking oligo (blocking oligo B2);
SEQ ID: NO 8, a 25-mer 3′-reference oligo (reference probe P2);
SEQ ID: NO 9, a target DNA with 2 5′-ATCT-3′ repeats;
SEQ ID: NO 10, a target DNA with 3 5′-ATCT-3′ repeats;
SEQ ID: NO 11, a target DNA with 4 5′-ATCT-3′ repeats;
SEQ ID: NO 12, a target DNA with 5 5′-ATCT-3′ repeats;
SEQ ID: NO 13, a target DNA with 6 5′-ATCT-3′ repeats;
SEQ ID: NO 14, a target DNA with 7 5′-ATCT-3′ repeats;
SEQ ID: NO 15, a target DNA with 8 5′-ATCT-3′ repeats;
SEQ ID: NO 16, a target DNA with 9 5′-ATCT-3′ repeats;
SEQ ID: NO 17, a target DNA with 10 5′-ATCT-3′ repeats;
SEQ ID: NO 18, a target DNA with 11 5′-ATCT-3′ repeats;
SEQ ID: NO 19, a target DNA with 12 5′-ATCT-3′ repeats;
SEQ ID: NO 20, a target DNA with 13 5′-ATCT-3′ repeats;
SEQ ID: NO 21, a target DNA with 14 5′-ATCT-3′ repeats;
SEQ ID: NO 22, a target DNA with 15 5′-ATCT-3′ repeats; and
SEQ ID: NO 23, a target DNA with 16 5′-ATCT-3′ repeats.
The mixtures or kits used to analyze any STR loci are very dependant on the sequence of the STR loci. But, to analyze one unknown STR locus, one would need to perform at least 3 experiments as follows:

Exp 1: two probes* (i.e. 16-mer, 20-mer) P1,P1′; one reference probe* P2, both flanking oligos F1,F2;
Exp. 2; two probes* (i.e. 16-mer, 20-mer) P1,P1′; one reference probe* P2, one blocking oligo B2 and one flanking oligo F1; or
Exp. 2′; two probes* (i.e. 16-mer, 20-mer) P1,P1′; one reference probe* P2, one blocking oligo B1 and one flanking oligo F2;
Exp. 3: two probes* (i.e. 16-mer, 20-mer) P1,P1′; one reference probe* P2, both blocking oligos B1,B2;

The asterix (*) refers to probes that contain a fluorescent label. Each label within a single experiment must be different. If an STR locus contains an insert (“island”), then all three experiments would also contain an insert probe P3, also labeled. More complex STRs might also require a third probe of a different length (also fluorescently labeled) and/or additional experiment(s) using different length blocker(s).
For distinguishing alleles of one particular STR, an example is given in FIG. 13. When inspecting FIG. 13, one should keep in mind, that all results are displayed as a ratio of the repeat signal (fluorescence intensity of bound STR probes P1) to the total DNA signal (fluorescence intensity of bound reference probes P2). For heterozygous samples however, the outputs would represent averages of the signals from each of the two alleles. This provides the opportunity for signals to be at half steps from the homozygous samples (i.e. 0.5, 1.5, 2.5, etc)
FIG. 13 represents the predicted probe signal ratios obtained from a set of experiments when the sample is not necessarily homozygous for STR repeat number. The table is explained as follows. We assume here that the sample examined will be biallelic as it is in human DNA, i.e. each of two copies of the examined STR locus have an independent number of STR repeats and are homozygous if the repeat numbers are equal or heterozygous if the repeat numbers are different. Across the top of the table are the number of repeats on allele 1 (5 through 16 in this example) and down the left side of the table are the number of repeats on allele 2 (also 5 through 16). Predicted probe signal ratios are displayed for a set of 6 measurements for each combination of alleles: either no blocker, 1 blocker, or 2 blockers are used in combination with either a 20-mer or 16-mer probe. The blockers in these experiments each can hybridize with and “block” with the 3 terminal repeats from either end of the repeat region (12 nucleotides worth). Thus for each combination of allele 1 and allele 2, 6 signal measurements are made, and the predicted ratio of these six signal measurements is shown on a horizontal in the table. For example, for the combination of 11 STR repeats (allele 1) and 15 STR repeats (allele 2), we would expect the signal ratios to be 2.5:2.5:1.5:2.5:1.0:1.5 for experiments with a 20-mer probe and no blocker, 16-mer probe and no blocker, 20-mer probe and one blocker, 16-mer probe and one blocker, 20-mer probe and two blockers, and 16-mer probe and two blockers, respectively. The underscored values on the diagonal in the table represent the homozygous condition where allele 1 and allele 2 have the same number of STR repeats. A unique pattern is still provided by each possible allelic combination.

Claims

What is claimed is:

1. A method of detecting the number of repeat units in a selected short tandem repeat (STR) in a genomic sample, the method comprising the steps of:

a) providing at least one:

a1) genomic sample containing the selected STR;

a2) set of polymerase chain reaction—(PCR) oligonucleotides for carrying out PCR amplification of the selected genomic sample with the short STR sequence;

a3) STR probe oligonucleotide (P1,P1′) with individual fluorescent label;

a4) reference probe oligonucleotide (P2) with a different fluorescent label;

b) amplifying the genomic sample with the selected STR sequence provided in step a1) by PCR using the set of PCR oligonucleotides provided in step a2), denaturing the amplified double stranded sample DNA for generating single stranded DNA, selecting and separating a single stranded target DNA for use in hybridization experiments;

c) carrying out partial genotyping by differential hybridization that comprises the steps:

c1) mixing an amount of the single stranded target DNA selected and separated in step b) with the STR probe oligonucleotides provided in step a3) and the reference probe oligonucleotide (P2), and allowing hybridization to the single stranded target DNA in a hybridization experiment;

c2) measuring the intensity of the fluorescence provided by the labeled STR probe oligonucleotides that are bound to the repeat units of the selected STR;

c3) measuring the intensity of the different fluorescence provided by the labeled reference probe oligonucleotide (P2) that is bound to a flanking sequence of the target DNA; and

c4) normalizing the intensity measured of the STR probe fluorescence in step c2) based on the intensity measured of the different fluorescence in step c3) and revealing a relative fluorescence signal representing the result of the differential hybridization experiment.

2. The method of claim 1,

wherein a first STR probe oligonucleotide (P1) is a 16-mer probe to a 5′-ATCT-3′ repeat.

3. The method of claim 1,

wherein in step a3) a second STR probe oligonucleotide (P1′) is provided in addition.

4. The method of claim 3,

wherein the second STR probe oligonucleotide (P1′) is a 20-mer probe to a 5′-ATCT-3′ repeat.

5. The method of claim 1, further comprising the step:

a5) providing a set of flanking oligonucleotides (F1,F2);

wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF).

6. The method of claim 1, further comprising the step:

a6) providing a blocking oligonucleotide (B2) and a flanking oligonucleotide (F1);

wherein in step c1), the blocking oligonucleotide (B2) and the flanking oligonucleotide (F1) provided in step a5) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a second differential hybridization experiment (FB).

7. The method of claim 1, further comprising the step:

a7) providing a blocking oligonucleotide (B1) and a flanking oligonucleotide (F2);

wherein in step c1), the blocking oligonucleotide (B1) and the flanking oligonucleotide (F2) provided in step a7) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in an alternative second differential hybridization experiment (BF).

8. The method of claim 1, further comprising the step:

a8) providing a set of blocking oligonucleotides (B1,B2);

wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB).

9. The method of claim 1, further comprising the steps of:

a5) providing a set of flanking oligonucleotides (F1,F2);

wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF);

wherein in step c1), the blocking oligonucleotide (B2) and the flanking oligonucleotide (F1) provided in step a5) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a second differential hybridization experiment (FB);

a8) providing a set of blocking oligonucleotides (B1,B2);

wherein in step c1), the set of blocking oligonucleotides (B1,B2) provided in step a8) is admixed to a first amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a third differential hybridization experiment (BB);

wherein the steps c2), c3), and c4) are carried out for the three differential hybridization experiments; and

wherein three relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,FB,BB) are achieved.

10. The method of claim 1, further comprising the steps of:

a5) providing a set of flanking oligonucleotides (F1,F2);

wherein in step c1), the blocking oligonucleotide (B1) and the flanking oligonucleotide (F2) provided in step a7) are admixed to a second amount of the single stranded target DNA, to a first STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in an alternative second differential hybridization experiment (BF);

a8) providing a set of blocking oligonucleotides (B1,B2);

wherein three relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,BF,BB) are achieved.

11. The method of claim 1, further comprising the steps of:

a5) providing a set of flanking oligonucleotides (F1,F2);

wherein in step c1), the set of flanking oligonucleotides (F1,F2) provided in step a5) is admixed to a first amount of the single stranded target DNA, to the a STR probe oligonucleotide (P1), and to the reference probe oligonucleotide (P2), and hybridization to the single stranded target DNA is allowed in a first differential hybridization experiment (FF);

a8) providing a set of blocking oligonucleotides (B1,B2);

wherein the steps c2), c3), and c4) are carried out for the four differential hybridization experiments; and

wherein four relative fluorescence signals representing the individual results of the three differential hybridization experiments (FF,FB,BF,BB) are achieved.

12. The method of claim 1,

wherein the reference probe (P2) is labeled with Cy5 at the 3′-end of the oligonucleotide.

13. The method of claim 2,

wherein the first STR probe (P1) is labeled with FAM at the 3′-end of the oligonucleotide.

14. The method of claim 3,

wherein the second STR probe (P1′) is labeled with Cy3 at the 3′-end of the oligonucleotide.

15. A kit (XX) for carrying out the method of partial genotyping by differential hybridization of claim 1, the kit (XX) comprising:

one STR probe oligonucleotide (P1); and

one reference probe oligonucleotide (P2).

16. A kit for carrying out the method of partial genotyping by differential hybridization of claim 3, the kit comprising:

one first STR probe oligonucleotide (P1);

one second STR probe oligonucleotide (P1′);

one reference probe oligonucleotide (P2); and one of:

two flanking oligonucleotides (F1,F2); or

one blocking oligo (B1) and one flanking oligo (F2); or

one flanking oligo (F1) and one blocking oligo (B2); or

two blocking oligonucleotides (B1,B2).

17. The kit of claim 16, further comprising:

one insert probe oligonucleotide (P3).

18. A kit (FF) for carrying out the method of partial genotyping by differential hybridization of claim 5, the kit (FF) comprising:

one STR probe oligonucleotide (P1);

one reference probe oligonucleotide (P2); and

two flanking oligonucleotides (F1,F2).

19. A kit (FB) for carrying out the method of partial genotyping by differential hybridization of claim 6, the kit (FB) comprising:

one STR probe oligonucleotide (P1);

one reference probe oligonucleotide (P2);

one flanking oligonucleotide (F1); and

one blocking oligonucleotide (B2).

20. A kit (BF) for carrying out the method of partial genotyping by differential hybridization of claim 7, the kit (BF) comprising:

one STR probe oligonucleotide (P1);

one reference probe oligonucleotide (P2);

one blocking oligonucleotide (B1); and

one flanking oligonucleotide (F2).

21. A kit (BB) for carrying out the method of partial genotyping by differential hybridization of claim 8, the kit (BB) comprising:

one STR probe oligonucleotide (P1);

one reference probe oligonucleotide (P2); and

two blocking oligonucleotides (B1,B2).