CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 61/235,999, filed August 21 , 2009, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology and more specifically to methods for molecular fingerprinting for the characterization and identification of organisms.

BACKGROUND OF THE INVENTION

Central to the field of microbiology is the ability to positively identify microorganisms at the level of genus, species, or serotype. Correct identification is not only an essential tool in the laboratory, but it plays a significant role in the control of microbial contamination in the processing of food stuffs, the production of agricultural products, and the monitoring of environmental media, such as ground water. Of greatest concern is the detection and control of pathogenic microorganisms. Typically, pathogen identification has relied on methods for distinguishing phenotypic aspects, such as growth or motility characteristics, and for immunological and serological characteristics. Selective growth procedures and immunological methods are the traditional methods of choice for bacterial identification and these can be effective for the presumptive detection of a large number of species within a particular genus. However, these methods are time consuming and are subject to error. Selective growth methods require culturing and subculturing in selective media, followed by subjective analysis by an experienced investigator. Immunological detection (e.g., ELISA) is more rapid and specific, however, it still requires growth of a significant population of organisms and isolation of the relevant antigens. For these reasons, interest has turned to detection of bacterial pathogens based on nucleic acid sequence.

Nucleic acid polymorphism provides a means to identify species, serotypes, strains, varieties, breeds, or individuals based on differences in their genetic make up. Nucleic acid polymorphism can be caused by nucleotide substitution, insertion, or deletion. The ability to determine genetic polymorphism has widespread application in areas such as genome mapping, genetic linkage studies, medical diagnosis, epidemiological studies, forensics, and agriculture. Several methods have been developed to compare homogenous segments of DNA to determine if polymorphism exists.

One method for determining genetic polymorphism uses primers of an arbitrary sequence to amplify DNA by the polymerase chain reaction (PCR) (Williams et al., Nucleic Acids Res. 18:6531-35 (1990); U.S. Patent No.

5,126,239, incorporated herein by reference). Because the primers are not designed to amplify a specific sequence, the technique is called random amplification of polymorphic DNA (RAPD) or arbitrarily primed PCR (APPCR). The primers used are at least seven nucleotides in length. Under the proper conditions, differences as small as a single nucleotide can affect the binding of the primer to the template DNA, thus resulting in differences in the distribution of amplification products produced between genomes.

Another method for identifying and mapping genetic polymorphisms has been termed amplified fragment length polymorphism (AFLP; U.S. Patent No. 5,874,215, incorporated herein by reference). AFLP combines the use of restriction enzymes with the use of PCR. Briefly, restriction fragments are produced by the digestion of genomic DNA with a single or a pair of restriction enzymes. If a pair of enzymes is used, enzymes are paired based on differences in the frequency of restriction sites in the genome, such that one of the restriction enzymes is a "frequent cutter" while the remaining enzyme is a "rare cutter." The use of two enzymes results in the production of single and double digestion fragments. Next, double stranded synthetic oligonucleotide adaptors of 10-30 bases are ligated onto the fragments generated. Primers are then designed based on the sequence of the adapters and the restriction site. When pairs of restriction enzymes are used, nucleotides extending into the restriction sites are added to the 3' end of the primers such that only fragments generated due to the action of both enzymes (double cut fragments) are amplified. Using this method, any polymorphism present at or near the restriction site will affect the binding of the primer and thus the distribution of the amplification products. In addition, any differences in the nucleotide sequence in the area flanked by the primers will also be detected. AFLP allows for the simultaneous co-amplification of multiple fragments.

A further method is Direct Linear Analysis (DLA), which analyzes individual DNA molecules bound with sequence-specific tags (see Chan et al., Genome Res. 14:1137-46 (2004); U.S. Patent No. 6,263,286, incorporated herein by reference). The method is intended to identify repetitive information in DNA, which is moved past at least one station, at which labelled units of DNA interact with the station to produce a DNA-dependent impulse. Because the extended objects are similar, or preferably identical, and comprise a similar, or preferably identical, pattern of labelled units, a characteristic signature of interactions is repeated as each extended object moves past a station or a plurality of stations. This repetitive information is extracted from the overall raw data by means of an autocorrelation function and is then used to determine structural information about the DNA.

Another method is amplification of repetitive elements (REP-PCR). This technique is based on families of repetitive DNA sequences present throughout the genome of diverse bacterial species (reviewed by Versalovic et a/., Methods MoL Cell. Biol. 5:25-40 (1994)). Repetitive extragenic palindromic (REP) sequences are thought to play an important role in the organization of the bacterial genome. Genomic organization is believed to be shaped by selection and the differential dispersion of these elements within the genome of closely related bacterial strains can be used to discriminate between strains (see, e.g., Louws et ai, Appl. Environ. Micro. 60:2286-95 (1994)). REP-PCR utilizes oligonucleotide primers complementary to these repetitive sequences to amplify the variably sized DNA fragments lying between them. The resulting products are separated by electrophoresis to establish the DNA "fingerprint" for each strain.

The output data of these fingerprinting systems generally is measured by assigning band sizes, though these assignments are somewhat imprecise depending on the sizing ladder used for the comparison. In addition, the output data can be difficult to compare between laboratories and often relies on the use of expensive proprietary software programs (such as BioNumerics, Applied Maths, Austin, TX) to handle the data.

SUMMARY OF THE INVENTION

Applicants have solved the aforementioned problems by embedding the 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 powerful, freeware bioinformatics tools.

One aspect is for a method of 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 of a DNA sequencing reaction 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.

In some aspects, the method comprises 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.

Another aspect is for an isolated polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

Other objects and advantages will become apparent to those skilled in the art upon reference to the detailed description that hereinafter follows.

SUMMARY OF THE SEQUENCES

SEQ ID NOs:1-4 and 25 are the nucleotide sequences of oligonucleotide primers useful in the present invention. Each primer can be employed alone or in conjunction with one or more other primers. For example, SEQ ID NOs:1-4 can be employed together to create the FB1 D1 primer mix, while SEQ ID NO:25 can be employed alone as the FP5 primer.

SEQ ID NOS:5-7, 13, and 14 are the nucleotide sequences resulting from operating the method of the present invention with negative control PCR reactions obtained using the FB1 D1 primer set.

SEQ ID NOS:8-12 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FB1 D1 primer set and Saccharomyces cerevisiae DNA.

SEQ ID NOS:15-19 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FB1 D1 primer set and Salmonella enterica DNA.

SEQ ID NOS:20-24 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FB1 D1 primer set and Staphylococcus aureus DNA.

SEQ ID NOS:26-30 are the nucleotide sequences resulting from operating the method of the present invention with negative control PCR reactions obtained using the FP5 primer. SEQ ID NOS:31-35 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FP5 primer and Staphylococcus aureus DNA.

SEQ ID NOS:36-40 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FP5 primer and Salmonella enterica DNA.

SEQ ID NOS:41-45 are the nucleotide sequences resulting from operating the method of the present invention with PCR reactions obtained using the FP5 primer and Saccharomyces cerevisiae DNA.

SEQ ID NO:46 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:21-23.

SEQ ID NO:47 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:33-35.

SEQ ID NO:48 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:9-11.

SEQ ID NO:49 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:41, 43, and 45.

SEQ ID NO:50 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:17-19.

SEQ ID NO:51 is the consensus nucleotide sequence obtained from a sequence comparison of SEQ ID NOS:36-38.

The sequences conform with 37 C.F.R. §§ 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid

Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-jb/s), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A shows a phylogram generated from a Clustal W alignment of all sequence reads from primer mix FB1 D1 of Example 1.

Figure 1B shows a phylogram generated from a Clustal W alignment of all sequence reads from single primer FP5 of Example 1.

DETAILED DESCRIPTION

Applicants specifically incorporate the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.

The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of."

The term "oligonucleotide" as used herein refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides.

The term "primer" as used herein refers to an oligonucleotide of any arbitrary sequence, whether occurring naturally, as in a purified restriction digest, or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. It is preferable that primers are sequences that do not form a secondary structure by base pairing with other copies of the primer or sequences that form a "hair pin" configuration. The sequence conveniently can be generated by computer or selected at random from a gene bank. The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an

oligodeoxyribonucleotide.

In the present disclosure, primers used for amplification based fingerprint methods are labelled with a fluor. Following generation of the fingerprint products by amplification, the fingerprint amplicons are comingled with the product of a previously performed DNA sequencing reaction. The comingled products are then run to produce a DNA sequence from a fluorescent DNA sequencing instrument. The sequence output is perturbed at positions where the fingerprint products are migrating with like-sized DNA sequencing fragments. The perturbations result in an altered DNA sequence output from the instrument. These alterations are reproducible, and comparison of the output sequences can be used to characterize and/or identify the organism whose DNA was subject to the fingerprinting method.

The nucleic acids to be analyzed by a process described herein may be DNA or RNA, and the DNA or RNA may be double stranded or single stranded. Any source of nucleic acid, in purified or nonpurified form, can be utilized as the starting nucleic acid. For example, the nucleic acid may be from a natural DNA or RNA from any source, including virus, bacteria, and higher organisms such as plants, animals, and microbes or from cloned DNA or RNA. Additionally, the nucleic acid may constitute the entire nucleic acid or may be a fraction of a complex mixture of nucleic acids. Preferably, the nucleic acid is deoxyribonucleic acid.

Processes described herein are applicable to any nucleic acid-containing starting material, including foods and allied products, vaccines and milk infected with a virus or a bacterium, whole blood, blood serum, buffy coat, urine, feces, liquor cerebrospinalis, sperm, saliva, tissues, and cell cultures (such as mammalian cell cultures and bacterial cultures). The processes are also applicable to relatively pure input materials, such as the product of a PCR or the product to be purified further of another process for recovering nucleic acids.

The step of generating an amplified nucleic acid product can be performed by, for example, RAPD PCR, AFLP PCR, REP-PCR, or DLA. Using RAPD as an example, the choice of nucleic acid polymerase used in the extension reaction, depends on the nature of the template. For DNA template strands, suitable commercially available DNA polymerase includes DNA polymerase obtained from the thermophilic bacterium Thermus aquaticus (Taq polymerase) or other thermostable polymerases. Structural variants and modified forms of this and other DNA polymerases would also be expected to be useful in the process of the present invention. For RNA templates, reverse transcriptase is an example of a DNA polymerase that would also be expected to be useful. In the presence of the nucleoside triphosphate substrates, natural or analogues, the polymerase extends the length of the primer in the 3' direction. The sequence of the extension product will generally be complementary to the corresponding sequence of the template strand.

The nucleoside triphosphate substrates are employed as described in PCR Protocols, A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. -J. Sninsky and T. J. White, eds. pp. 3-12, Academic Press (1989), which is incorporated by reference, and U.S. Patent Nos. 4,683,195 and 4,683,202, both incorporated by reference. The substrates can be modified for a variety of experimental purposes in ways known to those skilled in the art. As an example, at least one of the natural nucleoside triphosphate substrates may be replaced by a mobility-shifting analogue as taught in U.S. Patent No. 4,879,214, which is incorporated by reference.

Specifically, U.S. Patent No. 4,683,202 to Mullis is directed to a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. The process of Mullis comprises treating separate complementary strands of the nucleic acid with a molar excess of two

oligonucleotide primers, and extending the primers to form complementary primer extension products, which act as templates for synthesizing the desired nucleic acid sequence. The primers of Mullis are designed to be sufficiently complementary to different strands of each specific sequence to be amplified. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.

In one embodiment, at least one primer of greater than seven nucleotides is provided. Primers can be synthesized by standard techniques known to those skilled in the art. In some embodiments, at least one primer of nine to ten nucleotides in length is employed. Conveniently, one primer is employed. The at least one primer is labelled, preferably with a fluorophore, which can be, for example, dR6G, dR110, dTAMRA, dROX, VIC, NED, PET, LIZ, 6-FAM, TAMRA, DyeMer488/615, DyeMer488/630, PE-TexasRed, ECD, Alexa Fluor 610RPE, FITC, Oregon Green 488, or Qdot525. Other fluorophores can also be

employed.

In some embodiments, a nucleic acid is contacted with at least one oligonucleotide primer as described herein. The extension product is dissociated from the complementary random nucleic acid on which it was synthesized to produce a single-stranded molecule; and the random nucleic acid segment is amplified by contacting the single-stranded extension product with a primer from above under conditions as, for example, disclosed in PCR Protocols and U.S. Patent No. 4,683,202 such that an amplification extension product is synthesized using the single strand produced (i.e., the dissociated extension product) as a template.

The comingled products are then run to produce a DNA sequence from a fluorescent DNA sequencing instrument. The sequence output is perturbed at positions where the fingerprint products are migrating with like-sized DNA sequencing fragments. The perturbations result in an altered DNA sequence output from the instrument. These alterations are reproducible, and comparison of the output sequences can be used to characterize and/or identify the organism whose DNA was subject to the fingerprinting method using powerful freeware sequence analysis tools such as BLAST and Clustal W.

A process disclosed herein can be used to construct a nucleic acid 'fingerprint'. Such fingerprints are specific to individual organisms and can be applied to problems of identification or distinguishing of individual organisms. Such a fingerprint would be constructed using multiple polymorphisms generated by different primers and detected by the present invention, just as the

polymorphisms are used to create a fingerprint in Jeffreys, A. J., "Individual- Specific 'Fingerprints' of Human DNA", Nature 316:76-79 (1985), which is incorporated herein by reference. That is, genomes are compared for the presence of absence of polymorphisms.

In some embodiments, the steps of generating amplification products and producing an amplification profile after mixing the amplifications products with the oligonucleotide size ladder can be repeated at different stringency conditions as compared to that of a first pass through the process to generate a different amplification profile as compared to that generated by the first pass. Multiple repetitions are of course possible.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred

embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the preferred features of this invention, and without departing from the spirit and scope thereof, can make various changes and modification of the invention to adapt it to various uses and conditions.

Example 1

The hypothesis that labeled amplification based fingerprinting products could be detected and reproducibly placed within a DNA sequence by means of the invention was tested using Random amplification of polymorphic DNA (RAPD) fingerprinting to generate the fingerprinting products. PCR was performed using a mix of four primers labeled at the 5' end with a FA fluor, collectively known as primer mix FB1 D1 and single primer FP5 (see Table 1).

For primer mix FB1 D1 , each primer was present in the reaction at 0.25 μ concentration in the presence of other components necessary for performing polymerase chain reaction (nucleotides, polymerase, buffer) in a total reaction volume of 30 μΙ; for single primer FP5, it was present in the reaction at 0.1 μΜ concentration in a total reaction volume of 30 μΙ, in the presence of the other components required by polymerase chain reaction.

Reactions were run either with or without (negative controls) the addition of purified microbial DNA from three diverse organisms (one yeast, one gram positive bacterium and one gram negative bacterium (Table 2)) at a

concentration of 30 ng per reaction. Five replicates each were run for the negative control and each of the microbial DNA's.

PCR was carried out using a 2 minute hold at 95 °C followed by 10 cycles of 15 seconds at 95 °C, 5 minutes at 40 °C and 1 minute at 70 °C, followed by 30 cycles of 95 °C for 15 seconds and 3 minutes at 70 °C.

PCR reaction products were cleaned up as appropriate for DNA sequence reactions prior to loading on a capillary electrophoresis sequence apparatus, at which time the PCR products are recovered in a 15 μΙ volume of H ₂ 0.

A 2 μΙ aliquot of the PCR product is then added to 20 μΙ of deionized water. A commercial sequence standard (hsp 60, Applied Biosystems, Foster City, CA) is prepared as follows. A 1 μΙ aliquot of the sequence standard is mixed with 9 μΙ of formamide (HiDi, Applied Biosystems). 1.5 μΙ of the diluted PCR product is then added to the 10 μΙ sequence standard/formamide solution.

Samples are then mixed, denatured as for a standard sequencing reaction and loaded on to an Applied Biosystems 3730 DNA sequencer and run using standard DNA sequencing conditions. The output sequence files are then analyzed using standard DNA sequence analysis tools.

In order to test the ability of this invention to characterize an organism as belonging to a group (characterization) the sequences were examined using the Clustal W program (European Bioinformatics Institute web server). Two sets of alignments of sequences produced from primer mix FB1 D1 and single primer FP5 of Example 1 are shown in Tables 3A and 3B and the resulting phylograms are shown in Figure 1A and 1 B. As hypothesized, the perturbations of the sequence due to the comingling of the PCR products from the RAPD

fingerprinting reaction were detected as changes in the sequence output from the instrument. Clustal W alignments show that the replicate samples from a single organism cluster together and are separate from the clusters for non-identical microorganisms.

In order to test the ability of the invention to provide a means of identification by comparison to a database, the first three sequence embedded fingerprints (Numbers 1-3) generated for each microorganism were used to produce a consensus fingerprint sequence for that organism (Tables 4-6). These consensus sequences were then used to create a BLAST database (NCBI BLAST web server). The fifth sequence embedded fingerprint (number 5) for each organism was used to query the database. The resultant blast scores (Tables 7-9) show that the comparison of BLAST program identifies each microorganisms sequence embedded fingerprint as belonging to the correct species (highest total score).