Claims for Patent: 6,844,159

Summary for Patent: 6,844,159

Title:	Genetic markers for screening animals for improved disease resistance (NRAMP)
Abstract:	A method for determining improved innate immunity, disease resistance or performance in animals is disclosed. The method involves assays for a genetic differences in the NRAMP1 gene of the animal which is associated with superior disease resistance. Novel NRAMP1 sequence, assays, and compositions for identifying the presence of absence of these alleles are provided.
Inventor(s):	Tuggle; Christopher K. (Ames, IA), Marklund; Lena (Ames, IA), Stabel; Thomas J. (Ames, IA), Mellencamp; Martha A. (St. Joseph, MO), Stumbaugh; Amber (San Carlos, CA)
Assignee:	Iowa State University Research Foundation, Inc. (Ames, IA) Pig Inprovement Company UK Limited (GB) The United States of America as represented by the Secretary of Agriculture (Washington, DC)
Application Number:	10/160,948
Patent Claims:	1. A method of screening pigs to determine those more likely to exhibit a biological difference in disease resistance or innate immunity traits comprising: obtaining a sample of genetic material from said pig; and assaying for the presence of a genotype in said pig which is associated with a biological difference in disease resistance or innate immunity, said genotype characterized by the following: a) a polymorphism in the intron2/exon2 of the NRAMP1 gene. 2. The method of claim 1 wherein said polymorphism is located in a region of the NRAMP1 gene selected from the group consisting of: exon 15, intron 2/exon 2, the promoter region, and intron 1 or their equivalents as determined by a BLAST comparison. 3. The method of claim 1 wherein said polymorphism is identifiable by an Ava II restriction enzyme. 4. The method of claim 1 wherein said genotype is a Ava II polymorphism in intron 2/axon 2 of the NRAMP1 gene. 5. The method of claim 1 wherein said polymorphism results in an A⇄G transition in intron 2/exon 2 or its equivalent as determined by a BLAST comparison of SEQ ID NO:3. 6. The method of claim 1 wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). 7. The method of claim 1 further comprising the step of amplifying a region of intron2/exon2 of the NRAMP1 gene or a portion thereof which contains said polymorphism. 8. The method of claim 7 wherein said amplification includes the stops of: selecting a forward and a reverse sequence primer capable of amplifying said region of intron2/exon2 of the NRAMP1 gene which contains a polymorphic site. 9. The method of claim 8 wherein said forward and reverse primers are selected from SEQ ID NOS: 1 and 2. 10. The method of claim 7 wherein said amplification includes the steps of: selecting a forward and a reverse sequence primer capable of amplifying a region of the NRAMP1 gene which contains a polymorphic Ava II. 11. The method of claim 10 wherein said forward and reverse primers are selected from and based upon primer SEQ ID NO:1 and primer SEQ ID NO:2. 12. A method of screening pigs to determine those more likely to exhibit a biological difference in disease resistance or innate immunity traits comprising: obtaining a biological sample of material from said pig; and assaying for the presence of a genotype in said pig which is associated with a biological difference in disease resistance or innate immunity traits said genotype characterized by the following: a) a polymorphism in intron2/exon2 of the NRAMP1 gene, said polymorphism resulting in and characterized by a nucleotide at a position in intron2/exon2 of SEQ ID NO:3. 13. The method of claim 12 wherein said polymorphism results in an Ava II polymorphic restriction site. 14. A method for screening pigs to determine those with biologically different disease resistance or innate immunity traits, which method comprises of the steps: determining the alleles in intron2/exon2 of SEQ ID NO:3 of NRAMP1 present in a pig, said alleles having a polymorphic Ava II site in the NRAMP1 gene; determining the alleles of other markers for genes known to affect innate immunity; and selecting for a pig with a favorable combination of alleles associated with improved resistance to pathogenic infection observed when a favorable combination of alleles are present and against those not able to stave off infection when the favorable combination of alleles is not present. 15. The method of claim 14 wherein the determination of NRAMP1 alleles comprises determining the presence of at least one allele associated with at least one DNA marker linked either directly or indirectly to NRAMP1. 16. The method as claimed in claim 14 wherein the DNA marker is a microsatellite. 17. A method of identifying the presence or absence of an allele associated with a biological difference in disease resistance or innate immunity in a pig comprising: analyzing intron2/exon2 of SEQ ID NO:3 in the NRAMP1 gene encoding sequence present in said pig; comparing said sequence with the sequence of NRAMP1 alleles known to be associated with a biological difference in disease resistance or innate immunity and correlating said allele with the trait of a biological difference in disease resistance or innate immunity in a pig, a group or a population of pigs. 18. A method of screening pigs to determine those more likely to have biologically different general health traits comprising: obtaining a sample of genetic material from said pig; and assaying for the presence of a genotype in said pig which is associated with biologically different improved general health traits, said genotype characterized by the following: a) a polymorphism in intron2/exon2 of SEQ ID NO:3 in the NRAMP1 gene. 19. The method of claim 18 wherein said polymorphism is identifiable by an Ava II restriction enzyme. 20. The method of claim 18 wherein said genotype is a Ava II polymorphism in intron 2/exon 2 of the NRAMP1 gene. 21. The method of claim 18 wherein said polymorphism results in an A⇄G transition in intron 2/exon 2 or its equivalent as determined by a BLAST comparison of SEQ ID NO:3. 22. The method of claim 18 wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). 23. The method of claim 18 further comprising the step of amplifying region of intron2/exon2-of said NRAMP1 gene or a portion thereof which contains said polymorphism. 24. The method of claim 23 wherein said amplification includes the steps of: selecting a forward and a reverse sequence primer capable of amplifying said region of intron2/exon2 in the NRAMP1 gene which contains a polymorphic site. 25. The method of claim 24 wherein said forward and reverse primers are selected from SEQ ID NOS: 1 and 2. 26. The method of claim 23 wherein said amplification includes the steps of: selecting a forward and a reverse sequence primer capable of amplifying said region of the NRAMP1 gene which contains a polymorphic Ava II. 27. The method of claim 26 wherein said forward and reverse primers are selected from and based upon primer SEQ ID NO:1 and primer SEQ ID NO:2.

Details for Patent 6,844,159

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2021-05-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2021-05-31
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2021-05-31