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Table 1 Oligonucleotides for sequencing and genotyping Prnp alleles

From: Prevalence of the prion protein gene E211K variant in U.S. cattle

Primer numbera DNA sequenceb Gene region Orientation Function Tm (°C) STS length (bp) Termination mixture Mass (Daltons) Codons 210/211c
67661 gac att aga atc act tcc ata gg Intron 2, CDS, 3' UTR Sense Amplification, sequencing 58 1498 - d - -
65547 ata ctg agc taa cgg gac tt Intron 2, CDS, 3' UTR Antisense Amplification, sequencing 58 1498 - - -
65617 gat ttt tac atg ggc ata tga Intron 2, CDS, 3' UTR Sense Amplification, sequencing 60 985 - - -
42618 gcc aag ggt att agc ata ca Intron 2, CDS, 3' UTR Antisense Amplification, sequencing 60 985 - - -
42642 cct gga ggc aac cgt tat CDS Sense Sequencing 62 - - - -
27774 ctc ctg cca cat gct tca t CDS Antisense Sequencing 62 - - - -
58117 tac agg cca gtg gat cag ta CDS Sense Amplification 60 203 - - -
58119 gcc cct cgt tgg taa taa CDS Antisense Amplification 60 203 - - -
58118 acg ttg gat gta cag gcc agt gga tca gta CDS Sense Amplification for hME (with mass tag) 60 226 - 9311.10 -
58120 tgg acg ttg gat ggc ccc tcg ttg gta ata a CDS Antisense Amplification for hME (with mass tag) 60 226 - 9582.20 -
58141 caccaagggggagaacttcac-C-G-aaactgacatcaagatgat CDS Sense control template for cE211 allele 93 - - 12958.40 -
58142 atcatcttgatgtcagttt-C-G-gtgaagttctcccccttggtg CDS Antisense control template for cE211 allele 93 - - 12868.30 -
64161 caccaagggggagaacttcac-T-G-aaactgacatcaagatgat CDS Sense control template for tE211 allele 93 - - 12973.50 -
64162 atcatcttgatgtcagttt-C-A-gtgaagttctcccccttggtg CDS Antisense control template for tE211 allele 93 - - 12852.30 -
58143 caccaagggggagaacttcac-C-A-aaactgacatcaagatgat CDS Sense control template for cK211 allele 93 - - 12942.40 -
58144 atcatcttgatgtcagttt-T-G-gtgaagttctcccccttggtg CDS Antisense control template for cK211 allele 93 - - 12883.30 -
58145 caccaagggggagaacttcac-T-A-aaactgacatcaagatgat CDS Sense control template for tK211 allele 93 - - 12957.50 -
58146 atcatcttgatgtcagttt-T-A-gtgaagttctcccccttggtg CDS Antisense control template for tK211 allele 93 - - 12867.30 -
62792 caa ggg gga gaa ctt cac CDS Sense hME primer 58 - ddAe 5557.60 -
- caa ggg gga gaa ctt cac ca CDS Sense CA allele analyte - - ddA 6144.00f cK211
- caa ggg gga gaa ctt cac ta CDS Sense TA allele analyte - - ddA 6159.00f tK211
- caa ggg gga gaa ctt cac cga CDS Sense CGA allele analyte - - ddA 6473.20f cE211
- caa ggg gga gaa ctt cac tga CDS Sense TGA allele analyte - - ddA 6488.20f tE211
58148 atc atc ttg atg tca gtt t CDS Antisense hME primer 49 - ddC/ddGe 5783.80 -
- atc atc ttg atg tca gtt tc CDS Antisense C allele analyte - - ddC/ddG 6057.00 nE211
- atc atc ttg atg tca gtt ttg CDS Antisense TG allele analyte - - ddC/ddG 6401.20 cK211
- atc atc ttg atg tca gtt tta g CDS Antisense TAG allele analyte - - ddC/ddG 6714.40 tK211
  1. aUSMARC primer number; GenBank Accession number AY335912
  2. bPrimer sequences are listed 5' to 3'; primers 58118 and 58120 are analogous to primers 58117 and 58119, respectively, but with additional nucleotides on the 5' end (5'-acgttggatg-3' and 5'-tggacgttggatg-3', respectively) to shift their masses out of the analyte range.
  3. cnE211 refers to either cE211 or tE211 since the genotype for this analyte is ambiguous in the antisense direction.
  4. dNot applicable.
  5. eMixture also contains the complement dNTPs.
  6. fAlthough a conservative hME assay design includes 40 Dalton spacing between analytes, only 15 Dalton spacing was possible in this case. Nevertheless, the typical instrument resolution of approximately 4 Daltons is sufficient to resolve these analytes.