Kit for determining nucleic acid degradation

Abstract

A kit for use in assessing the status of nucleic acid degradation and/or the integrity of one or more nucleic acids in a sample by amplifying at least two overlapping regions within at least one locus and detecting the amount of the at least two amplification products. The kit includes a primer and at least two probes that bind under stringent conditions to a sequence that shares at least 80% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NO. 6 to SEQ ID NO. 47 over a stretch of 80 base pairs, or to a reverse complement thereof. One of the at least two probes binds to one of the at least two overlapping regions and the other of the at least two probes binds to a non-overlapping region.

Claims

1. A kit for use in assessing the status of nucleic acid degradation and/or the integrity of one or more nucleic acids in a sample by amplifying at least two overlapping regions within at least one locus and detecting the amount of the at least two amplification products, said kit comprising: a primer; and at least two probes that bind under stringent conditions to a sequence that shares at least 80% sequence identity to a sequence selected from the group of sequences consisting of SEQ ID NO. 6 to SEQ ID NO. 47 over a stretch of 80 base pairs, or to a reverse complement thereof; wherein one of said at least two probes binds to one of said at least two overlapping regions and the other of the at least two probes binds to a non-overlapping region.

2. The kit according to claim 1, wherein the primer and the at least two probes bind to a sequence selected from the group of sequences consisting of SEQ ID NO. 6 to SEQ ID NO. 47, or to a reverse complement thereof.

3. The kit according to claim 1, wherein the at least two probes have sequences that differ by no more than 5 nucleotides over a stretch of 20 nucleotides from SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

4. The kit according to claim 1, wherein the primer has a sequence that differs from a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 by no more than 5 nucleotides over a stretch of 20 nucleotides.

Description

EXAMPLES

(1) The following example are used in conjunction with the Figures and Tables to illustrate the invention.

(2) Description of Experiments for Obtaining the Results Depicted in FIGS. 4 and 5

(3) Environmental degradation may occur with forensic casework sample and is a typical challenge in routine genetic fingerprinting. Data presented here for the invention demonstrate the performance of the newly developed system for accurate DNA degradation detection.

(4) Male genomic DNA was sheared with a Covaris™ S220 Focused-ultrasonicator to an average fragment size of 1500 bp, 500 bp, 400 bp, 300 bp, 200 bp and 150 bp and 4.6 ng of each was tested with the invention for DNA degradation detection FIG. 4 (A).

(5) Shown in FIG. 4 (B) is U.S. Pat. No. 9,040,243 B2/EP 10178914 applied on the same subset of degraded DNA fragments. Depicted in FIGS. 4 (C) and (D) are commercially available kits from Supplier A and Supplier P, tested with the same subset and amount of fragmented DNA like in Figure (A).

(6) The commercially available quantification kits were set up and analyzed as described in the respective handbooks. A serial dilution of human DNAs (isolated from human blood from anonymous donors using the QIAamp Investigator Kit) and mixtures thereof at the concentrations described was used as a template for all of the kits tested.

FIGURE LEGEND

(7) FIG. 1: Two Genomic Regions Targeted on One Genomic Locus

(8) Depicted is a schematic drawing of the invention targeting different genomic regions on one genomic locus. The smaller PCR system consists of primer 1 and 2 and an appropriate probe for detection in qPCR and targets genomic region A. The larger PCR system consists of the same primer 1 and primer 3 extending the first genomic region A for the desired length and forming the second genomic region AB, which harbours the whole genomic region A.

(9) FIG. 2: Three Genomic Regions Targeted on One Genomic Locus

(10) Depicted is a schematic drawing of how the invention can be further extended targeting further genomic regions on one genomic locus. The smaller PCR system consists of primer 1 and 2 and an appropriate probe for detection in qPCR and targets genomic region A. The larger PCR system consists of the same primer 1 and a primer 3 extending the first genomic region A for the desired length and forming the second genomic region AB, which harbours the whole genomic region A. The larger PCR system consist of primer 1 and primer 4 extending genomic region A and genomic region AB and forming genomic region ABC and harbouring genomic region A and genomic region AB.

(11) FIG. 3: Schematic Drawings of State of the Art Solutions to Address DNA Degradation or Integrity

(12) A) Two Non-Overlapping Genomic Regions Targeted on One Genomic Locus

(13) FIG. 3 (A) shows a schematic drawing of targeting two genomic regions on a genomic locus, here a multi copy locus (Invention U.S. Pat. No. 9,040,243 B2, EP10178914). The smaller PCR system consists of an appropriate primerlprol5e system for detection in qPCR and targets genomic region D. The larger PCR system consists of an appropriate primer/probe system for detection in qPCR and targets genomic region E which is a separate but adjacent genomic region to genomic region D. Genomic region E does not harbour or overlap with genomic region D.

(14) B) Two Genomic Regions Targeted on Different Genomic Loci

(15) FIG. 3 (B) depicts an approach where different genomic regions from different genomic loci are targeted. The smaller PCR system consists of an appropriate primer/probe system targets genomic region F on genomic locus X. The larger PCR system consists of an appropriate primer/probe system that targets genomic region G on genomic locus Y.

(16) FIG. 4: Different Measurement Methods of Degraded DNA in Humans

(17) A) Method According to the Invention

(18) The invention shows no increase for the Ct values for the smallest PCR system (90 bp) for compromised DNA with an average fragment length from 1500 bp, 500 bp and 400 bp. Only for 300 bp, 200 bp and 150 bp there is an increase of up to 1.42 Ct values. Surprisingly the larger PCR system (352 bp) shows already a shift of 0.5 Ct when applied on fragmented DNA of 1500 bp length. Furthermore, the Ct values increase consistently on every further tested fragment length from 500 bp, 400 bp, 300 bp, 200 bp to 150 bp, and reach their maximum at 150 bp with more than 8 Ct values compared to undegraded DNA. This allows for a precise assessment of the degradation or integrity status of the DNA.

(19) B) Method According to U.S. Pat. No. 9,040,243B2/EP 10178914

(20) This graph shows the invention from U.S. Pat. No. 9,040,243 82/EP 10178914 applied on the same subset of degraded DNA fragments. The smallest PCR system (146 bp) shows already an increase for a fragment length of 1500 bp when compared to undegraded DNA. This difference increases with every further fragment length tested (500 bp, 400 bp, 300 bp, 200 bp and 150 bp). The longer PCR system (363 bp) shows an increase in Ct values when applied on the 1500 bp fragment length. The CT value increases further when the 500 bp fragment length was tested. For the 400 bp and 300 bp fragment lengths tested only a slightly increase in Ct values was observed while a detection for 200 bp fragments and 150 bp fragments completely failed.

(21) C) Method According to Supplier A

(22) Shown is a testing with the commercially available “Quantifiler® Trio DNA Quantification Kit” from Applied Biosystems/Thermo Scientific. Reactions were setup up according to manufacturer's handbook. The Quantifiler TRIO kit uses a small PCR system of 80 bp length and a larger PCR system of 214 bp length. When the kit was applied to the different fragment lengths only a slightly increase in Ct values was observed for the longer PCR system when used with the 1500 bp, 500 bp, 400 bp and 300 bp fragment lengths. Only for 200 bp and 150 bp fragments a significant increase in Ct values was observable. This limits dramatically the ability of the assay to assess the degradation status or integrity of the DNA tested.

(23) D) Method According to Supplier P

(24) Shown is a testing with the commercially available “PowerQuant™ System” from Promega. Reactions were setup up according to manufacturer's handbook. The PowerQuant™ System kit uses a small PCR system of 84 bp length and a larger PCR system of 294 bp length. When the kit was applied to the different fragment lengths a significant difference in Ct value increase was observed for the 1500 bp and 500 bp fragments and for the 200 bp and 150 bp fragments. However, the PCR systems fail to detect a significant difference between 500 bp, 400 bp and 300 bp.

(25) FIG. 5: Comparison of Degradation Indices Between Different PCR Systems

(26) Shown are the degradation indices (i.e. the ratio of the amount of short fragments vs. the amount of long fragments (human S/human L)) of the different systems tested.

(27) Noticeably, the method according to the invention (second column) obtains much higher indices, in particular for the small fragments (a value of almost 80) compared to the other systems. This indicates a much higher sensitivity for the detection of degraded DNA. Highlighted in grey, are the fragment sizes, for which the degradation status cannot be properly assessed anymore when using the other commercially available kits (light grey) or the RGQD system (dark grey).

(28) FIG. 6: Sequences

(29) Shown are sequences for primers, probes and amplicons according to the invention. For amplicon sequences, the primer binding sites are indicated with capital letters.