Categorization of DNA samples
The present application describes methods for accurate and cost-effective categorization of DNA samples into different types of in vitro generated DNA or different types of natural DNA such as from different tissues and/or physiological/pathological states. The invention achieves categorization by comparing “signal ratios” that are correlated to ratios of methylation levels at specific genomic loci, but does not rely on calculation of actual methylation levels at any genomic locus. Therefore the disclosed inventive method eliminates the requirement for external DNA species and controls, thereby simplifying and increasing the accuracy of the assay. The described inventive technology also enables performing the categorization of DNA together with DNA profiling in the same reaction, thereby allowing for concomitant categorization and determination of identity of the samples.
1. A method for categorizing a mammalian DNA sample as semen or non-semen, comprising:
(A) applying the DNA mammalian sample to digestion with at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease, to obtain restriction enzyme-treated DNA;
(B) co-amplifying at least two different genomic loci from the same digest of restriction enzyme-treated DNA in a single reaction mixture using at least two different locus-specific primer pairs, wherein at least one of the loci is a restriction locus differentially methylated between semen and non-semen DNA, thereby generating a specific amplification product for each locus of said at least two different genomic loci, wherein said co-amplifying comprises using fluorescent PCR;
(C) determining a signal intensity of each amplification product in said single reaction mixture;
(D) calculating a signal ratio between the signal intensities of amplification products of the at least two different genomic loci determined in step (C) in said single reaction mixture;
(E) providing a reference value representing a ratio between signal intensities of amplification products of said at least two different genomic loci in semen DNA;
(F) comparing the signal ratio to the reference value; and
(G) determining that the categorical source of the DNA sample is semen if the signal ratio matches the reference value,
wherein the restriction endonuclease is Hhai and the at least two different genomic loci comprise L91762 (SEQ ID NO: 39) and L68346 (SEQ ID NO: 40), and wherein the step of amplifying is performed using the following primers:
1. L91762 (forward GCAGCAGGCCGCGGAGAAG (FAM) (SEQ
ID NO: 67); reverse AGCAGCTGTGCCGGGCCAG(SEQ ID NO:
82));
and
2. L68346 (forward CAGCAACAGCACCCAGCTTG (JOE) (SEQ
ID NO: 68); reverse CACAGGCTCAGTCGCGGATC (SEQ ID
NO: 83)).
2. The method of claim 1 , wherein a signal ratio of L91762/L68346 that is between 0.04 to 0.53 is indicative of semen.
3. The method of claim 1 , wherein the at least two different genomic loci further comprise at least one of L50468 (SEQ ID NO: 41), L14432 (SEQ ID NO: 42), L30139 (SEQ ID NO: 45), L15952 (SEQ ID NO: 49), and L26688 (SEQ ID NO: 51).
4. The method of claim 3 , wherein the step of amplifying is performed further using at least one of the following pairs of primers:
3. L50468 (forward AGGAAACCTCAGTAGCAAAATTG (JOE)
(SEQ ID NO: 69); reverse GCGAGACTTTAGGTGTGCATC
(SEQ ID NO: 84));
4. L14432 (forward CGTAGGCTGCGGTGAGCTC (FAM) (SEQ
ID NO: 70); reverse GATCCATGCCCGCTGGGATG (SEQ ID
NO: 85));
5. L30139 (forward GAGAAGCGGGAGGATGAGAC (FAM) (SEQ
ID NO: 73); reverse GACAGTATGCTGATGCTTCTTG (SEQ ID
NO: 92);
6. L15952 (forward CTCCCTGATTTACGACAAGTTC (FAM)
(SEQ ID NO: 77); reverse GACAGTATGCTGATGCTTCTTG
(SEQ ID NO: 92);
and
7. L26688 (forward CGGACCAGATTGCTGGTCAC (JOE) (SEQ
ID NO: 79); reverse CGACCTTGCCAGATGTTTGAC (SEQ ID
NO: 94).
5. The method of claim 1 , wherein the at least two different genomic loci further comprise L76138 (SEQ ID NO: 48) and L26688 (SEQ ID NO: 51), and wherein a signal ratio of L76138/L26688 that is between 2.04-19.56 is indicative of semen.
6. The method of claim 1 , wherein step (E) further comprises providing a plurality of reference values representing signal intensity ratios in mixtures of semen and non-semen at various proportions; step (F) further comprises comparing the signal ratio to the plurality of reference values; and step (G) comprises determining that the categorical source of the DNA sample is a mixture of semen and non-semen if the signal ratio matches one of said plurality of reference values.
7. The method of claim 1 , wherein signal intensity is the amplification product's fluorescence level measured during capillary electrophoresis.
8. The method of claim 1 , wherein step (B) is performed by real-time PCR.
9. The method of claim 1 , further comprising simultaneously performing DNA profiling with the DNA categorization.
10. The method of claim 1 , wherein the steps of digesting and co-amplifying the DNA sample are performed in a single tube.
11. The method of claim 1 , wherein said single test tube comprises DNA template, digestion and amplification enzymes, buffer, primers designed to specifically amplify the at least two different genomic loci, and accessory ingredients.
12. The method of claim 1 , wherein said single test tube is closed and placed in a thermal cycler, where the single reaction takes place.
13. The method of claim 1 , wherein the at least two different genomic loci further comprise (1) a plurality of restriction loci and (2) a locus lacking a recognition sequence of the methylation-sensitive or methylation-dependent restriction endonuclease, and wherein step (D) comprises calculating a plurality of signal ratios, between the signal intensity of the amplification product of the locus lacking said recognition sequence and the signal intensities of each restriction locus, and wherein step (F) is repeated for each of said plurality of signal ratios.
14. The method of claim 13 , wherein step (D) further comprises calculating signal ratios between the signal intensity of the amplification product of each one of said plurality of restriction loci and the signal intensity of the amplification product of each of the remaining of said plurality of restriction loci.
15. The method of claim 13 , wherein step (D) comprises calculating a plurality of signal ratios, between the signal intensity of the amplification product of each one of said plurality of restriction loci and the signal intensity of the amplification product of each of the remaining of said plurality of restriction loci.
16. The method of claim 1 , wherein no standard curve from control DNA is generated and wherein no actual methylation level at any genomic locus is indicated by said method.
17. The method of claim 1 , wherein the DNA mammalian sample is a human DNA sample.