Analysis of multiple single nucleotide polymorphisms closely positioned in the ovine PRNPgene using linear fluorescent probes and melting curve analysis

Table 4 Predicted Tms of rare PRNP polymorphisms

Probe	Polymorphisms	Oligonucleotide sequence (5'-3')	Mean Tm	SD
136A2	A136	GCCTGCTCATGGCACTTCCC	61.6°C	0.13
136A2	V136	GCCTGCTCATGACACTTCCC	53.3°C	0.13
136A2	A136 & S138R	GCCTGCGCATGGCACTTCCC	57.3°C	0
136A2	V136 & S138R	GCCTGCGCATGACACTTCCC	47.3°C	0.13
136A2	A136 & S138N	GCCTGTTCATGGCACTTCCC	55.9°C	0.13
136A2	V136 & S138N	GCCTGTTCATGACACTTCCC	44.9°C	0.18
154H2	H154	CATGTTTTCATGATAGTAACGGTC	57.2°C	0.18
154H2	R154	CATGTTTTCACGATAGTAACGGTC	50.2°C	0.18
154H2	H154 & R151C	CATGTTTTCATGATAGTAACAGTC	51.4°C	0
154H2	R154 & R151C	CATGTTTTCACGATAGTAACAGTC	43.1°C	0
154H2	H154 & R151H	CATGTTTTCATGATAGTAATGGTC	52.7°C	0.13
154H2	R154 & R151H	CATGTTTTCACGATAGTAATGGTC	45.2°C	0.13
154H2	H154 & R151G	CATGTTTTCATGATAGTAACCGTC	51.1°C	0
154H2	R154 & R151G	CATGTTTTCACGATAGTAACCGTC	42.8°C	0
154H2	R154L	CATGTTTTCAAGATAGTAACGGTC	52.2°C	0.18
171R6	R171	TTCTGGTTACTATACCGATCCACTG	56.9°C	0
171R6	Q171	TTCTGGTTACTATACTGATCCACTG	50.5°C	0.27
171R6	H171	TTCTGGTTACTATAATGATCCACTG	45.2°C	0.18
171R6	R171 & Y172D	TTCTGGTTACTATCCCGATCCACTG	50.5°C	0.28
171R6	Q171 & Y172D	TTCTGGTTACTATCCTGATCCACTG	46.3°C	0.13
171R6	H171 & Y172D	TTCTGGTTACTATCATGATCCACTG	40.7°C	0.13
141C3	L141	CCAAAATGTATAAGAGGCCTGCTC	54.1°C	0.17
141C3	F141	CCAAAATGTATAAAAGGCCTGCTC	45.1°C	0.13
141C3	L141 & S138N	CCAAAATGTATAAGAGGCCTGTTC	49.4°C	0.13
141C3	F141 & S138N	CCAAAATGTATAAAAGGCCTGTTC	39.2°C	0.17
141C3	L141 & H143R	CCAAAACGTATAAGAGGCCTGCTC	54.9°C	0.13
141C3	F141 & H143R	CCAAAACGTATAAAAGGCCTGCTC	45.6°C	0.13

Complementary oligonucleotides were employed to predict the affect of rare polymorphisms on probe melting temperature. Six replicate analyses were performed for each target oligonucleotide. The mean Tm and standard deviation (SD) for melting peaks is presented. The nucleotides representing the polymorphisms in codons 136, 141, 154 and 171 are highlighted along with rare polymorphisms in neighbouring codons.

ISSN: 1471-2334