Claims for Patent: 9,441,278

Summary for Patent: 9,441,278

Title:	Genotyping method for use in cattle traceability and means thereof
Abstract:	The invention discloses means and methods for genotyping an individual head of cattle. Individual\'s DNA is genotyped utilizing the herein defined PCR SNaP-shot protocol. The protocol comprises two PCR steps where the first step (PCR1) includes adding primers (SEQ ID No. 1-30) and/or primers extended at their 5\' end with a common 10 base motif (ACGTTGGATG) to the PCR1 reaction. The second step (PCR2) includes adding extension primers (SEQ ID No. 31-45), and/or primers adjacent to corresponding specific SNPs. Further steps include producing amplicons from a PCR1 mixture comprising template DNA and the first primer set to yield PCR1 products, using PCR1 products as templates to a set of extension primers to yield PCR2 products. Size and color separation is achieved by adding tails of different lengths to the PCR2 primers. PCR2 products are separated and the results compared with SNP profiles from the databank to obtain matching.
Inventor(s):	Cahana; Aviv (Moshav Sitria, IL), Shirak; Andrey (Petach Tikva, IL), Karniol; Baruch (Bat Yam, IL), Skalsky; Yitzchak (Nitzan M.P. Evtach, IL), Weller; Joel Ira (Rehovot, IL), Ron; Micha (Nes Tziona, IL), Seroussi; Eyal (Sha\'arei Tikva, IL)
Assignee:	BACTOCHEM LTD. (Nes Ziona, IL) STATE OF ISRAEL, MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT, A.R.O.--VOLCANI CENTER (Beit Dagan, IL)
Application Number:	13/000,314
Patent Claims:	1. A method of genotyping an individual head of cattle comprising steps of: a. genotyping a DNA sample obtained from said individual wherein said genotyping utilizes PCR SNaPshot protocol, said protocol comprising a first PCR step (PCR1) and a second PCR step (PCR2), further wherein said PCR1 step includes adding a first primer set consisting of PCR1 primers of SEQ ID No. 1-30 to PCR1 reaction comprising said DNA sample to yield PCR1 products, and said PCR2 step includes adding extension primers consisting of PCR2 primers of SEQ ID No. 31-45 to yield PCR2 products, said genotyping is performed by multiplexing single nucleotide polymorphisms (SNPs) by amplifying in concert said PCR1 products in a single tube, followed by multiplexing said SNPs by amplifying in concert PCR2 products in the tube; b. producing amplicons from said PCR1 reaction comprising said DNA sample and said first primer set to yield said PCR1 products; c. using said PCR1 products as templates to said extension primers to yield said PCR 2 products; d. separating said PCR2 products on a capillary sequencer, thereby obtaining SNP profile results; e. recording said SNP profile results into a databank; and, f. comparing said SNP profile results with SNP profiles from said databank using data mining software to obtain matching profiles. 2. The method according to claim 1, wherein said step of multiplexing comprises selecting SNP markers by applying IPLEX software. 3. The method according to claim 1, wherein said step of selecting said PCR1 primers comprises using GeneBank sequence-files as input. 4. The method according to claim 1, wherein said method additionally comprises using said PCR1 products as templates to PCR2 primers and extending said PCR2 primers, with four fluorescent nucleotide-analogues in SNaPshot analysis. 5. The method according to claim 1, wherein said method additionally comprises modification of length by introducing primer tails of adenine tracts interrupted by cytosine nucleotides to the 5' end of said PCR2 primers such that a variety of extended PCR2 products are formed. 6. The method according to claim 1, wherein said method comprises steps of: forming said PCR2 products by adding poly-A tails of different lengths to the PCR2 primers to enable accurate size and color separation; and stabilizing the tails of said PCR2 primers by adding cytosine nucleotides to form poly-AC tails. 7. The method according to claim 1, wherein said method comprises reversibly tracing said head of cattle to an individual institution selected from the group consisting of slaughterhouse, refrigeration facility, meat packing plant, meat processing plant, meat shipping plant, livestock transporter, farmers organization, market, wholesaler, distributor, quarantine facility stock yard, farm, ranch, stud farm and pasture. 8. The method according to claim 1, wherein said method comprises reversibly tracing said individual head of cattle to an individual supermarket, butcher shop, restaurant or market. 9. The method according to claim 1, wherein said method is used in matching individual breaking table meat cuts to said individual cattle. 10. The method according to claim 1, wherein said method is used in matching retail meat cuts to individual cattle. 11. The method according to claim 1, wherein said method is used in matching fragments of meat cuts of a processed meat product to individual cattle, further wherein said meat product is selected from the group consisting of a hamburger, sausage and salami. 12. The method according to claim 1, wherein said method is used for full traceability of cattle from birth to consumption. 13. The method according to claim 1, wherein said method is used for partial traceability of cattle from birth to consumption. 14. The method according to claim 1, wherein said method is used for concise traceability of cattle from birth to consumption. 15. A package for genotyping individual head of cattle, said package comprising: a. a first premixed primer set consisting of SEQ ID No. 1-30, located in genomic proximity to SNPs, wherein said first primer set enables amplification in concert of 15 PCR1 products in a single tube from a PCR1 mixture comprising cattle template DNA and said first primer set; b. a set of premixed 5' extended tail primers consisting of SEQ ID No. 31-45, said tail primers configured to yield 15 PCR2 products in a single tube after annealing with said PCR1 products, said PCR2 products separable on a capillary sequencer, thereby providing SNP profile results for individual cattle; and, c. associated data mining software enabling matching of said SNP profile results. 16. The package according to claim 15, wherein said package is provided with means to record, store, process and match, by means of software, said SNP profile results, with a databank containing SNP profiles. 17. The package according to claim 16, wherein said package provides matching of said SNP profile results with said databank at an ID power ranging from about 1.11.times.10-8 for Limousin to about 2.3.times.10-10 for Holstein and/or Mmax ranging from about 900,822 to about 43,671,580 across breeds, enabling parental exclusion probabilities in all breeds, and detection of about 99.9% cases of incorrect parenthood, when both parental genotypes are available.

Details for Patent 9,441,278

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Insmed Incorporated	IPLEX	mecasermin rinfabate	Injection	021884	12/12/2005		2028-06-19