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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.