WO2006081612A1 - Tracking and identification - Google Patents
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- WO2006081612A1 WO2006081612A1 PCT/AU2006/000124 AU2006000124W WO2006081612A1 WO 2006081612 A1 WO2006081612 A1 WO 2006081612A1 AU 2006000124 W AU2006000124 W AU 2006000124W WO 2006081612 A1 WO2006081612 A1 WO 2006081612A1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K11/00—Marking of animals
- A01K11/001—Ear-tags
- A01K11/003—Ear-tags with means for taking tissue samples, e.g. for DNA analysis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K11/00—Marking of animals
- A01K11/001—Ear-tags
- A01K11/004—Ear-tags with electronic identification means, e.g. transponders
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K11/00—Marking of animals
- A01K11/006—Automatic identification systems for animals, e.g. electronic devices, transponders for animals
- A01K11/008—Automatic identification systems for animals, e.g. electronic devices, transponders for animals incorporating GPS
Definitions
- This invention relates to devices and systems to collect biological systems for example using DNACollectorTM (provisional patent owned by Gribbles Molecular Science - 2004904829). This invention also relates to utilising cellular material within or onto sampling devices for subsequent testing of sample for genetic analysis such as genetic identification within the sample. More particularly by DNA fingerprinting of cells enclosed for genetic identification.
- this invention relates to removal of such cells from sample device and subsequent analysis for identification.
- the invention also relates to obtaining genetic and other information from the sample, with incorporation of the information into a tag, such tags may be incorporated with/into animals, incorporated with samples for tracking, and other applications. Such information can be linked to additional information associated with the tag and may be held in a database form within or external to the tag.
- the invention also relates to and is particularly suitable for, but not limited to, collection and genetic identification of non-invasive samples from animals and the like.
- this invention relates to genetic identification of animals by utilising collection devices for the collection from saliva or other biological samples from animals such as cattle, the recovery and processing of said sample suitable for genetic analysis, genetic analysis including but not limited genetic identification by DNA fingerprinting allowing genetic identification of the specific animal, the transfer of identifying information to a tag or identification in the animal, and conformation that signal has been received.
- the invention relates to the improvement of identification and tracking of samples by combining tagging technologies with DNA and other identification ⁇ ethods
- such devices may also contain a variety of other information.
- An example of such an embodiment is the tracking of forensic samples to establish and ensure chain-of-custody; forensic samples are tagged with identification particulars from the case incorporated into the tag, then after subsequent processing the required information (including DNA) is then transferred back to the tag.
- tags may also incorporate features including: positioning devices such as GPS (global positioning systems) to ensure that samples cannot be misplaced as well as tracking movement of the sample; recording devices detailing which person processed each stage of the sample; location detectors which can maintain a log to determine the location of samples at all time. _
- a further non-limiting example of this embodiment is the labelling of pathology and other biological and non-biological samples.
- general practitioners may take a sample of blood and label the specimen with a tag.
- the tag is used to track the location of the sample during transport and processing etc, the tests performed on such samples, the results of such tests with subsequent transfer of test results in a format readily usable by the clinician.
- information linked to the tag may comprise of variety of forms including but not limited to genetic identification, disease status and predisposition, parentage, location and source of animal birth, health records (such as vaccination), livestock quality markers such as QTLs for wool and meat quality, etc.
- the present invention describes a method for processing a variety of samples for analysis including combinations of the steps of:
- Inserting cellular material into a collection device such a device may also compose of mobile and/or micro devices such as microfluidic devices and so called DNA-on-a- chip devices B. Recovery of cellular material from device
- the tag may be in a variety of formats e.g. active, passive etc and forms e.g. bolus, capsule, label, ear-tag etc as well as write- once, write-many including tamper-proof and tamper evident forms.
- F The tag transmitting or allowing a signal or through automatic data capture technology confirming whether or not the genetic information has been correctly received or otherwise.
- G The association of tags into or onto animals either before or after information
- the tag having the capacity to receive and robustly store genetic information signals or forms.
- the tag will be in a WORM (write once, read many) format.
- the information stored on the tag can be held in such a way to provide secure access rights for example that it cannot be deleted or modified without alerting a user.
- K The ability of capturing and or storing genetic information in a database form in a format capable for use by a mobile device. Such information transfer many be uni, bi or multidirectional as appropriate to the application.
- M transmitting a signal or via an automatic data capture device confirming whether or not the sample is authentic or otherwise.
- Blood has been the traditional method of sample collection for decades. This collection method was very time consuming and costly, primarily due to the fact that a veterinarian must be onsite during the whole bleed process. When herd sizes exceed several hundred animals, this collection process can take over several days.
- the cost of hiring a veterinarian can easily range between $150 - $300 for a standard working day. As a vet can normal process only approximately 100 - 200 animals per day, this means that that the average sized herd of 1000 animals may take up to five days to bleed, costing a significant amount of money and time. For example 5 days @ $300 per day is $1500 for 1000 animals is $1.50 per animal solely for vet fees.
- Blood must be placed directly into blood tubes containing EDTA buffer to inhibit coagulation during the bleed and transportation to the laboratory.
- the blood tubes must also be stored on ice before, during and after the bleeding of each animal, and must be maintained at
- Bloods must be sent by courier in a chilled condition i.e. 4 0 C.
- the tubes must be placed in foam racks, surrounded by plenty of packing material to reduce the risk of breakage. Due the temperature requirements most samples will be sent via air courier.
- Blood - The collected blood can be frozen at -20 degrees centigrade in either the tube it was delivered in or in a specialized freezer bag. Blood can be stored this way for a considerable period with minimal degradation.
- the extracted DNA can be stored indefinitely in 0.1 X TE solution.
- Hair sampling is now increasingly being performed as a collection method for DNA samples from livestock. Whilst hair samples can often be taken by producers themselves, eliminating the need for a veterinarian, the collection process is usually very messy, with significant risk of cross contamination. This contamination comes from the multiple usage of the same tools for the collection from different animals. Another problem with collecting hair for DNA extraction is that if it not performed properly, resultant DNA can be very hard and time consuming to extract with poor reliability. Normally the hair sample is placed loose in a bag or is fixed onto a sample card which is posted to the laboratory. This random placement of hair in the bag or card makes it difficult to acquire the correct amount of hair follicles without handling most of the sample, and therefore introducing outside contaminates.
- Hair samples can be collected and placed in an envelope for transport to the laboratory. However significant extra care must be taken to individually wrap each sample to eliminate the chance of hairs becoming free and contaminating other samples. These samples can all be transported at room temperature.
- the hair follicles will remain viable for a 1-2 years. However they are difficult to store due to their packaging. Often resulting in a filing cabinet system of loose bags and cards which is highly susceptible to future contamination and processing error.
- semen is a labour intensive process that must be carried out by veterinarian.
- a -wide variety of techniques can be used for semen collection varying from animal mounting artificial vaginas to rectal stimulation but all require specialist equipment and take a considerable amount of time.
- the semen must be stored at 4 degrees centigrade once the sample has been collected. It must be placed on ice, or better still in liquid nitrogen for transportation by a courier to the laboratory. Often semen must also be stored very carefully using slow-cooling to maintain cell viability, particularly if the sample is also to. be used for reproductive technologies.
- semen collection is a very expensive way of sampling male livestock. Normally costing in excess of $100 per animal, semen sampling is therefore available to only elite producers and animal studs due to its high cost.
- a single semen sample can be separated into multiple straws.
- Semen straws can be stored at -80 degrees for extended periods of time. They can be stored at lesser temperatures but degrade more quickly. Due to the relatively small size of the straw, many straws can be stored within a small area, but this size does limit the amount of
- Semen straws can be kept indefinitely at -80 degrees.
- a "genetic marker” is meant any locus or region of a genome.
- the genetic marker may be a coding or non-coding region of a genome.
- genetic markers may be coding regions of genes, non-coding regions of genes such as introns or promoters, or intervening sequences between genes such as those that include tandem repeat sequences, for example satellites, microsatellites and minisatellites, although without limitation thereto.
- Preferred genetic markers are highly polymorphic and display allelic variation between individuals and populations of individuals.
- preferred genetic markers are short tandem repeat sequences (STRs), such as are used in a variety of genotyping applications such as DNA fingerprinting, forensic DNA analysis, pre-implantation genetic analysis and fetal genotyping.
- STRs short tandem repeat sequences
- nucleic acid designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
- genetic marker information is produced, at least initially, by amplification of the genetic markers present in a nucleic acid sample obtained from one or more individuals.
- Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999), which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et a/., 1996, J. Am. Chem. Soc.
- PCR polymerase chain reaction
- LCR ligase chain reaction
- SDA strand displacement amplification
- RCR rolling circle replication
- nucleic acid sequence-based amplification as for example described by Sooknanan et a/., 1994, Biotechniques 17 1077, which is incorporated herein by reference; and Q-D replicase amplification as for example described by Tyagi et a/., 1996, Proc. Natl. Acad. Sci. USA 93 5395 which is incorporated herein by reference.
- a preferred nucleic acid sequence amplification technique is PCR.
- the skilled person will also be aware of still further variations of nucleic acid sequence amplification technology that may be useful in amplifying genetic markers for the purposes of genotyping. _
- an "amplification product refers to a nucleic acid product generated by a nucleic acid amplification technique.
- a “primer” is usually a single-stranded oligonucleotide, preferably having 12-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- the genetic markers are amplified by "multiplex PCR", which involves a reaction utilizing a plurality of different primer sets (for example, primers for CF and sex) to amplify a plurality of genetic markers so that simultaneous diagnoses can be performed.
- multiplex PCR produces a plurality of different sized products, thereby facilitating discrimination between genetic markers and allelic forms thereof.
- PCR reactions utilizing a single set of primers amplifying one specific fragment are referred to herein as a "singleplex PCR"
- a preferred PCR system is "fluorescent PCR”. This system uses fluorescent primers and an automated DNA sequencer to detect PCR product (Tracy & Mulcahy, 1991 ,Biotechniques 11
- Fluorescent PCR has improved both the accuracy and sensitivity of PCR for genotyping
- Fluorescent amplification products are electrophoresed using gel or capillary systems and pass a scanning laser beam, which induces the tagged amplification product to fluoresce.
- the DNA sequencer combined with appropriate software is generally known as a
- Fluorescent PCR is highly sensitive, approximately 1000 times more sensitive than conventional gel analysis, (Hattori et al., 1992, Electrophoresis 13 560-565.).
- An advantage of fluorescent PCR is that several primers can be multiplexed together since different fluorescent dyes can be simultaneously identified even if the amplification size product ranges overlap each other (Kimpton et al., 1993, supra). These different dyes allow identification of one amplification product from the others even if the product sizes are within 1-2 bp of each other. This method has been applied to multiplexing as many as fifteen sets of primers although relatively high amounts of DNA are required.
- Fluorescent PCR has already been successfully applied to genetic screening for cystic fibrosis (Cuckle et al., 1996 British Journal of Obstetrics and Gynaecology 103 795-799), Down syndrome (Peril et al., 1994), muscular dystrophies (Schwartz et al., 1992, American Journal of _ _
- Quantitative PCR is where the amount of PCR product from each allele is compared, allowing a calculation of the relative number of chromosomes. This method has been applied to the detection of trisomies by utilising fluorescent PCR with polymorphic small tandem repeats
- STRs Adinolfi et al., 1995, Bioessays 17 661-664. These DNA markers have unclear exact genomic function, are found throughout the genome. STRs can also be used to determine the origin of the extra chromosome and, if maternally derived, whether the extra chromosome is derived from meiosis I or meiosis Il (Kotzot et al., 1996, European Journal of Human Genetics 4 168-174).
- STR profiling the method of the invention may be particularly useful for the purposes of "DNA fingerprinting", otherwise referred to as STR profiling.
- STR amplification products are produced by fluorescent multiplex PCR as hereinbefore described
- DNA fingerprinting has been used by forensic science utilizing DNA markers. These STRs are similar to those used for trisomy detection. Their wide variation in length and their distribution between individuals makes STRs preferred genetic markers. In addition, their small size makes them more likely to survive degradation and allow PCR amplification. These STRs are used to build up a series of identifying markers which are then combined to determine the DNA 'fingerprint' (Zeigle et al., 1992, Genomics 14 1026-1031). "DNA fingerprinting”, otherwise referred to as STR profiling.
- STR amplification products are produced by fluorescent multiplex PCR as hereinbefore described
- the STR profiling system has several advantages over alternative earlier methods (Jeffreys et al., 1985, Nature 316 76-79) using single locus probes (SLPs). It is more sensitive and requires only ⁇ 1ng of DNA compared to upwards of 50ng for SLPs. It can also be used for highly degraded DNA as it amplifies 100-400 bp compared to the 1,000-20,000bp lengths produced by SLPs. It can be performed in a single tube; Southern blotting or hybridisation is not required; and since alleles are discrete and can be sized precisely, the binding of alleles, a necessity in SLP analysis, is not required.
- amplification failure is where a genetic marker fails to be amplified.
- the reasons for amplification failure of genetic markers obtained from single cells are unclear but are likely to be numerous. They may include problems with sample preparation; e.g. failure to transfer the cell, degradation or loss of the target sequence and/or problems associated with the PCR.
- the major cause of PCR failure however is probably due to inefficient _ _
- PCR amplification failure occurs in the region of 15-30% of single cells (Li et al., 1988. Nature 335 414-419; Holding & Monk, 1989,
- allelic dropout also known as allele dropout
- allele dropout is failure to amplify one of two heterozygous alleles or the failure of one allele to reach the threshold of detection (preferential amplification).
- allelic dropout has been considered, for example, in microsatellite-based detection of cancers (reviewed by Cawkwell et al., 1995, Gastroenterology 109 465-471).
- allelic dropout increase appears to be inversely proportional to the amount of template in the sample and directly proportional to the number of primers contained in the PCR.
- allelic dropout rate 25%-33% in cells from heterozygote human embryos (Ray & Handyside, 1994 Miami Bio/Technology Short Reports: proceedings of the 1994 Miami Bio/Technology European symposium Advances in Gene Technology: Molecular Biology and Human Genetic Disease 5 46.).
- allelic dropout remains controversial as although most groups describe allele dropout, since some groups have reported no allelic dropout even in large numbers of single cell analyses (Verlinsky & Kuliev, 1992 Preimplantation diagnosis of genetic disease: A new technique in assisted reproduction. Wiley-Liss, New York.; Strom et al., 1994, Journal of Assisted Reproduction and Genetics 11 55-62.). In general though, the concept of allele specific PCR failure in single cells is relevant. _ _
- locus dropout is where neither allele is amplified to a detectable level.
- preferential amplification is the failure to amplify one allele of a heterozygous pair of alleles to reach a threshold of detection. In other words, one allele is amplified preferentially over another.
- fluorescent PCR is an ideal system to identify preferential amplification for two reasons. Firstly, it provides highly accurate and reliable detection of signals even when signal strength is very weak or many times lower (to ⁇ 1%) than the other allele. Secondly, it is quantitative. It is possible to use these quantitative measurements to accurately determine the ratio of signal intensity between the two alleles and thus determine the degree of preferential amplification.
- Amelogenin is a sex marker and a highly conserved gene (for tooth protein) found on both the X and Y chromosome, but is 6 base pairs longer on the Y chromosome (step 5). If the sample is male (with both X and Y) there will be a result of two peaks of 106bp (for gene on X chromosome) and 112bp (gene on Y chromosome); a female (2 copies of X) results in a single peak at 106bp.
- multiplex amplification or “multiplex PCR” refers to amplification of a plurality of genetic markers in a single amplification reaction.
- STR preferred genetic markers
- SNP markers preferred genetic markers.
- MFPCR methodology e.g. International Application PCT/AU02/01388 to successfully amplify multiple STR markers from limiting amounts of nucleic acid template.
- nucleic acid sequence amplification is not limited to PCR. - I -
- Nucleic acid amplification techniques are well known to the skilled addressee, and also include ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et a/. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999); strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et a/., 1996, J. Am. Chem. Soc.
- LCR ligase chain reaction
- SDA strand displacement amplification
- RCR rolling circle replication
- nucleic acid sequence-based amplification as for example described by Sooknanan et a/., 1994, Biotechniques 17 1077; and Q-D replicase amplification as for example described by Tyagi et a/., 1996, Proc. Natl. Acad. Sci. USA 93 5395.
- NASBA nucleic acid sequence-based amplification
- Q-D replicase amplification as for example described by Tyagi et a/., 1996, Proc. Natl. Acad. Sci. USA 93 5395.
- the abovementioned are examples of nucleic acid sequence amplification techniques but are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.
- an "amplification product' refers to a nucleic acid product generated by a nucleic acid amplification technique.
- nucleic acid other than DNA, preferably the nucleic acid is DNA.
- the nucleic acid is genomic DNA.
- SNP Single Nucleotide Polymorphisms
- SNP genotyping has multiple applications such as predictive medicine, personal medicine, forensic identification and pharmacogenomics. SNP genotyping has already been used to investigate a number of disorders such as cystic fibrosis, Factor V Leiden mutation, and factors such as A, B, O and Rh blood grouping.
- Preferred sources of nucleic acids are mammals.
- the invention particularly contemplates genetic analysis of human and non-human samples such as from cows, sheep, horses, pigs and any other mammal including companion animals, sporting animals and livestock, although without limitation thereto. Genetic identification is usually undertaken on samples which contain plentiful amounts of robust DNA such as blood, tissue and bacterial samples etc. Such samples are relatively easy to amplify as they usually contain many thousands of cells and often only involve a single test. _
- DNA fingerprints cannot be rubbed off, smudged, interfered with, or obscured.
- DNA fingerprints cannot be duplicated, manufactured or modified.
- DNA fingerprints can be obtained even after death unlike signatures or physical fingerprints. In fact, the inventors have demonstrated that DNA fingerprints can be obtained from samples many thousands of years old.
- a "genetic marker” is meant any locus or region of a genome.
- the genetic marker may be a coding or non-coding region of a genome.
- genetic markers may be coding regions of genes, non-coding regions of genes such as introns or promoters, or intervening sequences between genes such as those that include tandem repeat sequences, for example satellites, microsatellites and minisatellites, although without limitation thereto.
- Preferred genetic markers are highly polymorphic and display allelic variation between individuals and populations of individuals.
- preferred genetic markers are short tandem repeat sequences (STRs) 1 such as are used in a variety of genotyping applications such as DNA fingerprinting, forensic DNA analysis, pre-implantation genetic analysis and genotyping.
- STRs short tandem repeat sequences
- nucleic acid designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
- genetic marker information is produced, at least initially, by amplification of the genetic markers present in a nucleic acid sample obtained from one or more individuals.
- Nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) and ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et a/. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
- SDA strand displacement amplification
- RCR rolling circle replication
- nucleic acid sequence-based amplification as for example described by
- a preferred nucleic acid sequence amplification technique is PCR.
- the skilled person will also be aware of still further variations of nucleic acid sequence amplification technology that may be useful in amplifying genetic markers for the purposes of genotyping.
- an "amplification product refers to a nucleic acid product generated by a nucleic acid amplification technique.
- a “primer” is usually a single-stranded oligonucleotide, preferably having 12-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- the genetic markers are amplified by "multiplex PCR", which involves a reaction utilizing a plurality of different primer sets (for example, primers for CF and sex) to amplify a plurality of genetic markers so that simultaneous diagnoses can be performed.
- multiplex PCR produces a plurality of different sized products, thereby facilitating discrimination between genetic markers and allelic forms thereof.
- PCR reactions utilizing a single set of primers amplifying one specific fragment are referred to herein as a "singleplex PCR"
- a preferred PCR system is "fluorescent PCR”. This system uses fluorescent primers and an automated DNA sequencer to detect PCR product (Tracy & Mulcahy, 1991 , Biotechniques
- Fluorescent PCR has improved both the accuracy and sensitivity of PCR for genotyping (Ziegle et al., 1992, Genomics, 14 1026-1031 ; Kimpton et ai, 1993, PCR Methods and Applications 3 13-22).
- Fluorescent amplification products are electrophoresed using gel or capillary systems and pass a scanning laser beam, which induces the tagged amplification product to fluoresce.
- the DNA sequencer combined with appropriate software is generally known as a "Genescanner”. Stored data can then be analysed to provide product sizes and the relative amount of amplification product in each sample.
- Fluorescent PCR is highly sensitive, approximately 1000 times more sensitive than conventional gel analysis, (Hattori et al., 1992, Electrophoresis 13 560-565.).
- isolated or “isolation” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native or recombinant form.
- nucleic acid' designates single-or double-stranded mRNA, RNA, cRNA and DNA 1 said DNA inclusive of cDNA and genomic DNA.
- a "polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides.
- a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
- nucleic acid sequence amplification techniques are not presented as an exhaustive list of techniques. Persons skilled in the art will be well 10 aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.
- a preferred nucleic acid sequence amplification technique is PCR.
- an "amplification product refers to a nucleic acid product generated by a nucleic acid amplification technique.
- L5 A "primer” is usually a single-stranded oligonucleotide, preferably having 12-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- tag and “tag device” are used interchangeably and broadly cover microchips, automated data capture/storage devices, RFIDs, mobile RF transmitters and receivers, biometric devices, including but not limited to devices containing some or all or the
- JO following features devices commonly know as RFID devices, automated storage/capture devices, coil, chips, bolus etc, active devices, active or passive devices, devices using single or multiple frequencies such as VHF, UHF, devices incorporating multiple formats for example such as bar coding and RFID.
- Radio Frequency Identification RFID can minimize or eliminate many of the bar code
- RFID technology enables automatic and easier identification of objects remotely through the use of radio frequencies. Additionally, microchips are more suited for livestock environmental conditions. RFIDs exist in an array of sizes and forms. In one particular use, they are used for attachment and/or implantation onto/into animals e.g. glass capsule for injection, _
- This invention contemplates the use and application of many forms of such devices based upon the suitability of the device for each particular application.
- microchips are generally tiny, passive electronic devices, ranging in size from 12 to 28 millimeters in length and 2.1 to 3.5 millimeters in diameter. The smallest microchip is about the size of a grain of rice. During the manufacturing process, each microchip is individually inscribed and programmed to store a unique, permanent, alphanumeric identification code which identifies the tag.
- the microchip In current practice, once the animal has been identified with an RFID device, the microchip remains inactive until read with a scanner.
- the scanner sends a radio frequency signal to the chip, providing the power needed by the microchip to send its unique code back to the scanner and positively identify the animal using the identification code.
- the microchip is designed to last for the life of the animal with the unique ID number intact.
- a prize pig can be tagged then later taken to market, the tag can be removed and transferred to an inferior pig to realise an inflated price.
- Another example is of prize bulls being tagged, the tag being removed and transferred to another bull when the prize bull is too old or unavailable for stud, thus obtaining a higher stud fee for the inferior animal
- Yet another example is that of companion animals such as dogs and cats.
- a tag can be put into the animal and used for identification if the animal is lost or used for show, again the tag can be removed and transferred to another animal.
- BSE Breast disease tracking
- animals can be tracked and certified as coming from BSE free herds, tags can be removed and transferred to infected animals with the subsequent result of the meat entering the human food chain.
- the inventors realised this significant limitation and developed simple methods for using the animals individual DNA identification as a marker system for identification and subsequent tracking.
- tags for document security, for example of verification of documents.
- authentication or “verification” is meant to render authentic; to give authority to, to prove, to attest as being genuine, or sufficient to entitle to credit or the process of determining whether or not a set of established requirements are met.
- document is meant an item that 1. contains information that a user can view or understand; 2. anything serving as a representation of a person's thinking by means of symbolic marks; 3. writing that provides information (especially information of an official nature) or 4. a written account of ownership or obligation.
- the object of the invention is to provide a system where animals can be genetically identified with high degrees of specificity using tags, where the information within the tags is derived from the animals own genetic profile.
- a further object is to provide cost-effective animal tracking system.
- a further object of the invention is to streamline sampling collection; processing, analysis and animal labelling enabling much more cost effective and wide ranging testing.
- non-invasive testing for example detailed in patent application 2004904829 which minimises the requirement for a veterinarian, due to invasive nature of conventional sampling such as blood, hair etc, to be onsite for the collection process;
- a yet further specific object of the invention is to allow producers of farm animals to take samples from all herds for genetic analysis such as but not limited to genetic identification by DNA fingerprinting, with a minimum of time, effort and cost.
- Nucleic acid testing including for identification, genetic traits (including but not limited to tenderness, milk production etc), genetic disorders, disease and pathogen status.
- E. Tagging device capable of receiving such information in a readily accessible format e.g. WORM, read-write, EPROM device
- Tag or Data storage device capable of receiving verification data, usable data and security data in a readily accessible yet secure format.
- Inserting cellular material into a collection device may also compose of mobile and/or micro devices such as microfluidic devices and so called DNA-on-a- chip devices
- the tag may be in a variety of formats e.g. active, passive etc and forms e.g. bolus, capsule, label, ear-tag etc as well as write- once, write-many including tamper-proof and tamper evident forms.
- the tag transmitting or allowing a signal or through automatic data capture technology confirming whether or not the genetic information has been correctly received or otherwise.
- T The association of tags into or onto animals either before or after information (including genetic) transfer where appropriate.
- the tag having the capacity to receive and robustly store genetic information signals or forms.
- the tag will be in a WORM (write once, read many) format.
- the information stored on the tag can be held in such a way to provide secure access rights for example that it cannot be deleted or modified without alerting a user.
- X The ability of capturing and or storing genetic information in a database form in a format capable for use by a mobile device. Such information transfer many be uni, bi or multidirectional as appropriate to the application.
- the invention generally provides a method of generating identifying information for a biological material, the method including the steps of:
- the biological system includes one or more of the following biological organisms:
- the identifying information includes at least one of the following:
- the biological material includes:
- the sample is derived from:
- the biological organism is:
- the biological organism is a mammal.
- the mammal is a human.
- the mammal may be a non-human mammal.
- the step of collecting the sample of the biological system includes:
- the step of collecting the sample may include collecting either a:
- the method includes collecting a sample of, or from, hair, horn, nail, feathers or skin from the biological system.
- the method is non-invasive to the biological system.
- non-invasive it is meant that the performance of the method, neither the epidermis, nor any external bodily structure on the mammal is punctured or ruptured in the performance of the method. Accordingly, it will be understood that the following are examples of “non-invasive” procedures, within the meaning of that term as used throughout this specification: _
- the method includes the step of collecting a quantity of a biological fluid containing cells from the biological system.
- the biological fluid may include one or more of the following:
- the term "bodily discharges” includes: (a) genital discharges;
- the step of conducting an analysis on the sample of the biological material includes the use of a sampling device.
- the sampling device includes one or more of the following features:
- the device is designed so as to maintain the integrity of the sample
- the device is designed to maintain the integrity of the genetic information contained in the sample
- the device is designed to optimize the yield of the sample collected
- the device is designed to have a detachable segment that contains the sample
- the device is designed to identify the sample
- the device includes means to indicate whether a sample has been taken (for example, the device may include an indicator or colour formation means to identify when the sample has been taken);
- the device is designed to prevent or minimize the potential for contamination from other sources;
- the device is designed to prevent tampering with the sample, or to indicate attempts to tamper with the sample, or actual tampering.
- the step of conducting an analysis on the sample of the biological material includes an enrichment or isolation procedure, in order to obtain or derive one or more target materials from the sample.
- the target material may be:
- the enrichment or isolation procedure is either:
- the enrichment or isolation procedure includes one or more of the following:
- the step of conducting an analysis on the biological material contained in the sample includes the use of one or more of the following:
- the method includes one or more of the following:
- the results of the performance of the method are analysed.
- the step of transferring a copy or details of the identifying information to an information storage and/or retrieval system includes one or more of the following:
- the identifying information includes one or more of the following: (a) a quantity of one or more cells contained in the sample;
- the information derived from conducting an analysis in the sample is capable of being stored and/or retrieved: (a) in electronic form;
- the information is stored: (a) in accordance with sub-paragraphs (a) or (b) of the preceding paragraph, on an electronic information storage and/or retrieval means; and/or
- the information is stored:
- the step of transferring a copy or details of the identifying information to an information storage and/or retrieval system includes one or more of the following:
- the object is a mammal.
- the device is preferably:
- step of transferring a copy or details of the identifying information to an information storage and/or retrieval system is an invasive procedure, and includes one or more of the following:
- the object contains or includes paper, and the identifying information is affixed to or embedded in or linked to or associated with the object.
- the object may be any of the following: (a) a passport for international travel
- the invention additionally provides a method of identifying an individual, including the steps of:
- the individual is preferably a mammal.
- the mammal may be either a human or a non-human mammal.
- the step of transferring a copy or details of the identifying information to an information storage and/or retrieval system includes storing the identifying information in: _ _
- the method by which the identifying information is stored on the mammal by affixing the information to, or embedding the information in the mammal includes:
- the step of identifying the mammal includes using a reading means to detect the identifying information stored in or on the mammal.
- the reading means is a means capable of reading identifying information stored on one or more of the following:
- the individual is a human.
- the identifying information about the individual is stored in or on an object.
- the object may be any of the following:
- the invention yet further provides a method of verifying the authenticity of an object, including the steps of: (a) generating identifying information from a biological system in accordance with the method aspect of the invention;
- the object is preferably:
- L 5 (d) a credit or electronic commerce card or other device for securely performing a financial or other transaction where the identity of an authorised user of the card or device requires verification; and/or (e) an object to which rights of access or use need to be authenticated by reading identifying information contained on the object; .0 (f) an object which contains medical or other biological information about the biological system from which the identifying information was derived.
- the invention additionally provides a method of limiting rights to the use of an object, including the steps of:
- the identifying information can be read or retrieved using a reading means, so as to verify the presence of the identifying information in or on the object;
- the invention also provides an apparatus for generating identifying information for a biological material, the apparatus being suitable for use in accordance with one or more of the method aspects of the invention.
- the invention yet further provides an apparatus for identifying an individual, the apparatus being suitable for use in accordance with one or more of the method aspects of the invention.
- the invention also provides an apparatus for verifying the authenticity of an object, the apparatus being suitable for use in accordance with one or more of the method aspects of the invention.
- the invention yet further provides an apparatus for limiting the rights to use of an object, the apparatus being suitable for use in accordance with one or more of the method aspects of the L 5 invention.
- the invention additionally provides forms of apparatus suitable for use in performing one or more of the method aspects of the invention, in which the apparatus takes the form of a kit.
- sampling systems can be utilised depending upon the aspect of the invention for example, the tagging process and type of tag could vary from animal to animal.
- the invention relates to devices and systems to collect biological systems from animals for example using DNACollectorTM (provisional patent owned by Gribbles .5 Molecular Science - 2004904829).
- DNACollectorTM provisional patent owned by Gribbles .5 Molecular Science - 2004904829.
- Many animals including but not limited to, cattle, dogs and sheep continually produce excess amounts of saliva. This saliva, which normally escapes through the side of the mouth, contains cellular material and thus DNA from the oral cavity. In addition, some animals also produce significant amounts of mucus from the nasal cavity.
- One aspect of this invention is effectively a modified improved sampling device allowing 30 reliable and accurate sampling of such samples whilst maintaining subsequent stability of the genetic material.
- this invention relates to the processing of biological samples for forensic chain-of-custody applications.
- the format of such forensic samples may be in test-tubes, specimen containers such as tubes, small plastic containers known as eppendorf tubes, paraffin-embedded, plastinated or containers.
- specimen containers such as tubes
- small plastic containers known as eppendorf tubes
- paraffin-embedded plastinated or containers.
- the invention relates to the tracking of biological samples, a non- limiting example is samples taken from medical practitioners for medical testing. Such samples are again taken in a variety of formats such as collection tubes and forms and consist of a range of material including blood, semen, faeces, hair sputum, biopsy samples etc. However it will be 10 appreciated that this invention has application to almost any type or format of biological sample. Tagging and RFID Tagging
- RFIDs exist in an array of sizes and forms and are well known in the art. In one particular use, they are used for attachment and/or implantation onto/into animals e.g. glass
- L5 capsule for injection, ear tag, coil, chip, gut bolus etc.
- This invention contemplates the use and application of many forms of such devices based upon the suitability of the device for each particular application.
- genetic analysis and “genetic diagnosis” are used interchangeably and broadly cover detection, analysis, identification and/or characterization of isolated and nonisolated genetic material and includes and encompasses terms such as, but not limited to, genetic identification, genetic diagnosis, genetic screening, genotyping and DNA fingerprinting >5 (also commonly known as STR profiling) which are variously used throughout this specification.
- nucleic acid' designates single-or double-stranded mRNA, RNA, cRNA, RNAi and DNA inclusive of cDNA, genomic DNA and DNA-RNA hybrids.
- a “polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide” has less than eighty (80) contiguous nucleotides. >0
- a “SNP” is a single nucleotide polymorphism.
- a “primer” is usually a single-stranded oligonucleotide, preferably having 12-50 contiguous nucleotides which, for example, is capable of annealing to a complementary nucleic acid "template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM. !5
- a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
- marker or “marker” is meant any locus or region of a genome.
- the genetic marker may be a coding or non-coding region of a genome.
- genetic markers may be coding regions of genes, non-coding regions of genes such as introns or promoters, or intervening sequences between genes such as those that include polymorphisms (such as - OD - single nucleotide polymorphisms), tandem repeat sequences, for example satellites, microsatellites, short tandem repeats (STRs) and minisatellites, although without limitation thereto.
- polymorphisms such as - OD - single nucleotide polymorphisms
- tandem repeat sequences for example satellites, microsatellites, short tandem repeats (STRs) and minisatellites, although without limitation thereto.
- a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
- FISH fluorescence in situ hybridization
- PRINS primed in situ synthesis
- MFPCR multiplex fluorescent PCR amplification
- FISH fluorescent in situ hybridization
- PRINS Primed In situ Synthesis
- This invention also envisages increasing nucleic acid yield using high throughput amplification of nucleic acid product from pooled or limited samples such as single cells using generic kits such as Genomiphi (Amersham Bioscience).
- multiplex amplification or “multiplex PCR” refers to amplification of a plurality of genetic markers in a single amplification reaction.
- STR and/or SNP markers are preferred genetic markers. These is an extensive range of STR markers and primers (including International Application PCT/AU02/01388) together with MFPCR methodology to successfully amplify multiple STR markers from limiting amounts of nucleic acid template.
- nucleic acid sequence amplification is not limited to PCR.
- Nucleic acid amplification techniques are well known to the skilled addressee, and also include ligase chain reaction (LCR) as for example described in Chapter 15 of Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons NY, 1995-1999); strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252; rolling circle replication (RCR) as for example described in Liu et al., 1996, J. Am. Chem. Soc. _ _
- LCR ligase chain reaction
- SDA strand displacement amplification
- RCR rolling circle replication
- nucleic acid sequence-based amplification as for example described by Sooknanan et a/., 1994, Biotechniques 17 1077; and Q-D replicase amplification as for example described by Tyagi et a/., 1996, Proc. Natl. Acad. Sci. USA 93 5395.
- NASBA nucleic acid sequence-based amplification
- Q-D replicase amplification as for example described by Tyagi et a/., 1996, Proc. Natl. Acad. Sci. USA 93 5395.
- the abovementioned are examples of nucleic acid sequence amplification techniques but are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.
- an "amplification product' refers to a nucleic acid product generated by a nucleic acid amplification technique.
- nucleic acid other than DNA, preferably the nucleic acid is DNA.
- the nucleic acid is genomic DNA.
- SNP Single Nucleotide Polymorphisms
- SNP genotyping has multiple applications such as predictive medicine, personal medicine, forensic identification and pharmacogenomics. SNP genotyping has already been used to investigate a number of disorders such as cystic fibrosis, Factor V Leiden mutation, and factors such as A, B, O and Rh blood grouping.
- conventional SNP analysis is limited by the relatively high amount of extracted DNA usually required (up to 100ng) for analysis.
- genomic analysis there is increasing demand to both maximize data by performing multiple analyses and secondly to analyse minimum amounts of sample, even to the single cell level.
- multiple SNP analyses can be performed routinely, the degree if sensitivity is still far from single cell level analysis. Multiplex single cell SNP analysis has been problematic and again is not amenable to the high throughput processing required of clinical application.
- Preferred sources of nucleic acids are mammals.
- the invention also contemplates genetic analysis of samples such as from cows, sheep, horses, pigs and any other mammal including companion animals, sporting animals and livestock, although without limitation thereto. _
- GPS Global Positioning System
- GPS was developed by the United States Department of Defense as a military locating utility. However, over the past several years, GPS has also proven to be a useful tool in non-military mapping applications. GPS satellites orbit high enough to avoid the problems associated with land based systems, yet can provide accurate positioning 24 hours a day, anywhere in the world. Uncorrected positions determined from GPS satellite signals produce accuracies in the range of 50 to 100 meters. When using a technique called differential correction, users can get positions accurate to within 5 meters or less. As GPS units are becoming smaller and less expensive, there are an expanding number of applications for GPS.
- GPS assists pilots and drivers in pinpointing their locations and avoiding collisions.
- farmers can use GPS to guide equipment and control accurate distribution of fertilizers and other chemicals.
- GPS is used for providing accurate locations and as a navigation tool for hikers, hunters and boaters.
- the satellites and the receivers use very accurate clocks which are synchronized so that they generate the same code at exactly the same time.
- the code received from the satellite can be compared with the code generated by the receiver.
- the time difference between when the satellite generated the code and when the receiver generated the code can be determined. This interval is the travel time of the code. Multiplying this travel time, in seconds, by 186,000 miles per second gives the distance from the receiver position to the satellite in miles.
- GPS receivers There are three types of GPS receivers, which are available in today's marketplace. Each of the three types offers different levels of accuracy, and has different requirements to obtain those accuracies: Coarse Acquisition (C/A code) and Carrier Phase receivers and Dual Frequency receivers.
- C/A code Coarse Acquisition
- Carrier Phase receivers and Dual Frequency receivers.
- C/A Code receivers typically provide 1-5 meter GPS position accuracy with differential correction.
- C/A Code GPS receivers provide a sufficient degree of accuracy to make them useful in most GIS applications.
- C/A Code receivers can provide 1-5 meter GPS position accuracy with an occupation time of 1 second. Longer occupation times (up to 3 minutes) will provide GPS position accuracies consistently within 1-3 meters. Recent advances in GPS receiver design will now allow a C/A Code receiver to provide sub-meter accuracy, down to 30 cm.
- Carrier Phase receivers typically provide 10-30 cm GPS position accuracy with differential correction. Carrier Phase receivers provide the higher level of accuracy demanded by certain GIS applications. Carrier Phase receivers measure the distance from the receiver to the satellites by counting the number of waves that carry the C/A Code signal. This method of determining position is much more accurate; however, it does require a substantially higher occupation time to attain 10-30 cm accuracy. Initializing a Carrier Phase GPS job on a known point requires an occupation time of about 5 minutes. Initializing a Carrier Phase GPS job on an unknown point requires an occupation time of about 30-40 minutes. Additional requirements, such as maintaining the same satellite constellation throughout the job, performance under canopy and the need to be very close to a base station, limit the applicability of Carrier Phase GPS receivers to many GIS applications.
- Dual-Frequency receivers are capable of providing sub-centimeter GPS position accuracy with differential correction. Dual-Frequency receivers provide "survey grade" accuracies not often required for GIS applications. Dual-Frequency receivers receive signals from the satellites on two frequencies simultaneously. Receiving GPS signals on two frequencies simultaneously allows the receiver to determine very precise positions.
- This invention incorporates location detection systems such as GPS into tags, allowing tracking of tags at all times with very high degrees of accuracy.
- the applications herein also envisage use with RFID tags where there is a key structure that enables access and thus protects the data stored on the tag and also in other cases where the tag is an open platform tag that is able to be read by all who have a reader / writer working in accordance with the protocol of the tag.
- the tag is an open platform tag that is able to be read by all who have a reader / writer working in accordance with the protocol of the tag.
- some parts of the a tag may be configured to be read within the open standard and other parts of the tag read in accordance with a protected format.
- the information to be stored on the tag has been processed and determined, the information is converted to a format suitable to be stored on the tag.
- One particular advantage of using DNA based identification information is that this information renders the tag an effectively unique, yet readily verifiable marker system.
- the protection/security method, data transfer method and form and format the information are stored in, the information is stored within the RFID Tag with the level of protection/security and data transfer method the platform of the device uses.
- Reference samples either obtained by cellular material or known results from previously analysed nucleic acid samples can be directly compared to results obtained from nucleic acid results obtained from documents. Such results can be digitised or coded for example into allele numbering systems as is well known in the art, for computer implementation for rapid analysis and matching.
- the sample can be regarded as authentic or verified.
- This invention also envisages that samples could be subject to periodic and random inspections by testing authorities (such as MLA) or other authorities can occur to ensure DNA /
- Sampling and tag tracking can be incorporated as an entirely new development or alternatively be developed to work within existing tagging framework.
- DNA sample taken Sample referencing that Unique tag id - encrypted file copied to RFID smart label that's applied to sample.
- DNA Marker file sent back I to farmerVia email / GPRS / CD Copy etc. For uploading to tag
- One aspect of this invention is effectively a modified improved sampling device allowing reliable and accurate sampling of such samples whilst maintaining subsequent stability of the genetic material.
- a saliva/mucus sample can be collected easily and cheaply from each animal. Samples can be obtained during other on-farm treatments, such as branding and dipping, which results in significant time and cost savings.
- the saliva/mucus sample can be collected from either the mouth or nose region of the animal. This sample is then place in an individually label tube containing a stabilizing buffer for transport back to the lab for analysis.
- the sampling device also incorporates a colour indicator, which indicates to the collector whether if a sample has been taken correctly, for example by colour change.
- the tube that contains the sampling device incorporates sections for recording the animals' data, plus a barcode system for ease of identification.
- samples can be sent by domestic post with no additional care or packaging thus minimising requirement for expensive storage and transport requirements such as ice or liquid nitrogen.
- the samples are very stable at room/ambient temperature, and can even tolerate variations of heat (environmental conditions). Both the sampling device and tube are stable for extended periods of time, thus ensuring viable DNA analysis in the future.
- the sampling device may be as tamperproof as possible.
- the sampling device should also be tamper evident to indicate tampering and this minimise the risk of accepting contaminated samples as genuine.
- Nucleic acid may be obtained from a wide variety of cellular material such as skin scrapings, blood, buccal cells or other body fluids or samples. More preferably from noninvasive samples such as saliva or cervical samples for ease of use.
- the head is removed from the sampling device and transferred to a tamper-proof container, which contains further stabilising agents such as, but not limited to, phosphate buffered saline for several minutes to create a mixture allowing the sample to the retained in a robust state for maximum periods of time.
- the tamper-proof container is sealed using incorporated tamperproof and tamper evident adhesive.
- adhesive may contain light or gas activated agents, which indicate tampering.
- the sample is then stored, preferably in a dark area until required. Expensive storage and transport requirements such as ice or liquid nitrogen are not required.
- the cellular mixture would at this stage be dried onto the inserted material (such as paper or cloth).
- Nucleic acid can be removed from inserted material using a variety of methods.
- a volume of suspension fluid such as phosphate buffered saline
- Another non-limiting example is to remove the material from the container. The container is then agitated for several seconds to allow cellular material to be released into the container; the contents are then processed for genetic amplification.
- An advantage of fluorescent PCR is that several primers can be multiplexed together since different fluorescent dyes can be simultaneously identified even if the amplification size product ranges overlap each other (Kimpton et al., 1993, supra). These different dyes allow identification of one amplification product from the others even if the product sizes are within 1-2 bp of each other. This method has been applied to multiplexing as many as fifteen sets of primers although relatively high amounts of DNA are required.
- Fluorescent PCR has already been successfully applied to genetic screening for a variety of disorders such as cystic fibrosis (Cuckle et al., 1996 British Journal of Obstetrics and Gynaecology 103 795-799), Down syndrome (Peril et al., 1994), muscular dystrophies (Schwartz et al., 1992, American Journal of Human Genetics 51 721-729; Mansfield et al., 1993a. Human Molecular Genetics 2 43-50) and Lesch-Nyhan disease (Mansfield et al., 1993b. Molecular and Cellular Probes 7 311-324).
- STRs polymorphic small tandem repeats
- STR profiling for genetic identification.
- STR amplification products are produced by fluorescent multiplex PCR as hereinbefore described - -
- DNA fingerprinting for genetic identification has been used by forensic science utilizing DNA markers. These STRs are similar to those used for trisomy detection. Their wide variation in length and their distribution between individuals makes STRs preferred genetic markers. In addition, their small size makes them more likely to survive degradation and allow PCR 5 amplification. These STRs are used to build up a series of identifying markers which are then combined to determine the DNA 'fingerprint' (Zeigle et al., 1992, Genomics 14 1026-1031).
- STR profiling otherwise referred to as STR profiling.
- STR amplification products are produced by fluorescent multiplex PCR as hereinbefore described
- SLPs single locus probes
- amplification failure is where a genetic marker fails to be amplified.
- allelic dropout is failure to amplify one J5 of two heterozygous alleles or the failure of one allele to reach the threshold of detection (preferential amplification).
- Potential problems with the diagnosis of heterozygous individuals using PCR include the possibility of total amplification failure of one of the two heterozygous alleles whilst the other allele successfully amplifies (allelic dropout), or the failure of one allele to reach the threshold of detection (preferential amplification).
- allelic dropout has been considered, for example, in microsatellite-based detection of cancers (reviewed by Cawkwell et al., 1995, Gastroenterology 109 465-471).
- the rate of allelic dropout increase appears to be inversely proportional to the amount of template in the sample and directly proportional to the number of primers contained in the PCR.
- allelic dropout remains controversial as although most groups describe allele dropout, since some groups have reported no allelic dropout even in large numbers of single cell analyses (Verlinsky & Kuliev, 1992 Preimplantation diagnosis of genetic disease: A new technique in assisted reproduction. Wiley-Liss, New York.; Strom et al., 1994, Journal of Assisted Reproduction and Genetics 11 55-62.). In general though, the concept of allele specific PCR failure in single cells is relevant.
- locus dropout is where neither allele is amplified to a detectable level.
- preferential amplification is the failure to amplify one allele of a heterozygous pair of alleles to reach a threshold of detection. In other words, one allele is amplified preferentially over another.
- PCR is an ideal system to identify preferential amplification for two reasons. Firstly, it provides highly accurate and reliable detection of signals even when signal strength is very weak or many times lower (to ⁇ 1%) than the other allele. Secondly, it is quantitative. It is possible to use these quantitative measurements to accurately determine the ratio of signal intensity between the two alleles and thus determine the degree of preferential amplification. Differences in signal intensity in sister alleles can be either preferential amplification or allelic dropout. If the PCR produced allelic dropout rather than preferential amplification, no signal would be obtained with either technique and misdiagnosis of a carrier cell would occur. . .
- Amelogenin is a sex marker and a highly conserved gene (for tooth protein) found on both the X and Y chromosome, but is 6 base pairs longer on the Y chromosome (step 5). If the sample is male (with both X and Y) there will be a result of two peaks of 106bp (for gene on X chromosome) and 112bp (gene on Y chromosome); a female (2 copies of X) results in a single peak at 106bp.
- Reference samples either obtained by cellular material or known results from previously analysed nucleic acid samples can be directly compared to results obtained from nucleic acid results obtained from documents. Such results can be digitised for example into allele numbering systems as is well known in the art, for computer implementation for rapid analysis and matching. If a match, then the sample can be regarded as authentic or verified.
- Tag sample and unique identifier n iow linked. Tag transferred to sample. GPS system tracks sample within lab.
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