Genetic fingerprinting and identification method
The present disclosure provides methods for molecular fingerprinting for the characterization and identification of organisms. More specifically, in one aspect the present invention provides a method of identifying an organism in a sample by embedding fingerprint bands from any amplification based fingerprinting method within a DNA sequence so that small differences in size are resolvable. Fingerprint output is provided in a text file format that can then be analyzed by bioinformatics tools.
1. A method of generating a sequence embedded fingerprint pattern for identifying an organism in a sample comprising:
(a) providing a sample comprising said organism, said organism comprising at least one nucleic acid;
(b) combining said sample or the at least one nucleic acid therefrom with an amplification mix comprising at least one labeled oligonucleotide primer;
(c) generating at least one labeled amplification product from the at least one nucleic acid of said organism using a nucleotide amplification technique employing said at least one labeled oligonucleotide primer;
(d) combining said at least one labeled amplification product with products from a previously performed DNA sequencing reaction of a known organism to create a separation mix; and
(e) separating said separation mix on the basis of oligonucleotide length in a fluorescent DNA sequencing instrument to generate a sequence embedded fingerprint pattern for said organism, wherein the sequence embedded fingerprint pattern is generated by perturbing sequence output at positions where fingerprint products are migrating with like-sized DNA sequence fragments.
2. The method of claim 1 , comprising after step (e) the further steps of:
(f) comparing said sequence embedded fingerprint pattern for said organism to a database containing sequence embedded fingerprint patterns for known organisms; and
(g) identifying said organism as a function of said comparison to said database.
3. The method of claim 1 , wherein said organism is a microorganism.
4. The method of claim 1 , wherein said organism is unknown.
5. The method of claim 1 , wherein said nucleotide amplification technique is a random amplification of polymorphic DNA (RAPD) polymerase chain reaction (PCR).
6. The method of claim 1 , wherein said at least one nucleic acid is DNA.
7. The method of claim 1 , comprising after step (e) the further step of repeating steps (c)-(e) at different stringency conditions as compared to that of a first pass through steps (c)-(e) to generate a different amplification profile as compared to that generated by the first pass through steps (c)-(e).
8. The method of claim 1 , wherein step (e) is performed by capillary gel electrophoresis.
9. The method of claim 1 , wherein steps (b)-(c) are performed by one or more primers comprising nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:25, and a combination thereof.