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 utilising the herein defined PCR SNaPshot 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 colour 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.

1 .- 42 . (canceled) 
     
     
         43 . A set of DNA primers comprising: (a) a primer set consisting of SEQ ID No.1-30 located in genetic proximity to specific SNPs within cattle DNA; and (b) a set of 5′ extended stabilized PCR2 primers consisting of SEQ ID No. 31-45, wherein said SEQ ID No.1-30 primers provide multiple PCR1 products in a single tube from a PCR1 mixture; said mixture also comprising said cattle DNA; further wherein said PCR2 primers anneal to said PCR1 products and are comprising 5′ extended poly-AC tail PCR2 primers, and are further extended with four fluorescent nucleotides, which are analogues to the nucleotides adenine, cytosine, thymine and guanine, to form PCR2 products during SNaPshot analysis, said PCR2 products separable on a capillary sequencer.