1) Design of the APEX Primers for Detection of Single Base Substitution

1) Design of the APEX primers for detection of single base substitution

Mutation p.Met390Arg (NM_024649.4:c.1169T>G) in BBS1 gene.

Sense strand oligo: 5’GCGGGAGGACAACACCCTCATCA 3’

Expected wild type signal is T

Expected mutant type signal is G

c.1169T>G

GCGGGAGGACAACACCCTCATCA

1138 CGGTACGGGCGGGAGGACAACACCCTCATCATGACCACTCGAGgtgagtggagtcagacctg

Antisense strand oligo: 5’ GACTCCACTCACCTCGAGTGGTC 3’

Expected wild type signal is A

Expected mutant type signal is C

c.1169T>G

1138 CGGTACGGGCGGGAGGACAACACCCTCATCATGACCACTCGAGgtgagtggagtcagacctg

CTGGTGAGCTCcactcacctcag

All APEX signals were detected with Genorama QuattroImager and analysed using Genorama Genotyping Software 4.5. Given illustrations are taken from PicDB- Genorama Genotyping Software 4.5.

In the first row cells from left to right:

Fluorescent image in channels A,C,G and T respectively; Histograms represent relative signal intensities; BC-detected signal; in cells A,C,G,T ‘Histogra’ & BC top row corresponds to sense strand, bottom row- antisense strand.

Row 1. DNA sample has normal genotype for posit. c.1169. We have detected in sense strand normal T allele and in antisense strand we have detected normal A allele (T/A).

Row 2. DNA sample is heterozygous for c.1169T>G. We have detected in sense strand normal T allele and mutant G allele. In antisense strand we have detected normal A allele and mutant C allele (TG/AC).

2) Design of the APEX primers for detection of insertion

Mutation p.Cys91SerfsX5 (NM_024685.3:c.271dupT)in BBS10 gene.

Sense strand oligo: 5’GGTGCAAAAACATTTATTATCTTTCTTT 3’

Expected wild type signal is G

Expected mutant type signal is T

c.271dupT

GGTGCAAAAACATTTATTATCTTTCTTT

241 GATGGTGCAAAAACATTTATTATCTTTCTTTTGCCATTTGCTTAGAGGACTTCATGCAATC 300

Antisense strand oligo: 5’ ATGAAGTCCTCTAAGCAAATGGCAAA 3’

Expected wild type signal is G

Expected mutant type signal is A

c.271dupT

241 GATGGTGCAAAAACATTTATTATCTTTCTTTTGCCATTTGCTTAGAGGACTTCATGCAATC 300

AAACGGTAAACGAATCTCCTGAAGTA

Row 1. DNA sample has normal genotype for posit. c. 271. We have detected in sense strand normal G allele and in antisense strand we have detected normal G allele (G/G).

Row 2. DNA sample is heterozygous for c. 271dupT. We have detected in sense strand normal G allele and mutant T allele. In antisense strand we have detected normal G allele and mutant A allele (GT/GA).

3) Design of the APEX primers for detection of deletion

Mutation p.Val707X (NM_024685.3:c.2118_2119delTG) in BBS10 gene.

Sense strand oligo: 5’ AATATTAACCATTGACATGGTAATCACT 3’

Expected wild type signal is G

Expected mutant type signal is T

c.2118_2119delTG

AATATTAACCATTGACATGGTAATCACT

2087 CAAAAATATTAACCATTGACATGGTAATCACTGTTAAGAGACACCCTCAGAAAGTTCACAATCAA 2151

Antisense strand oligo: 5’ GTGAACTTTCTGAGGGTGTCTCTTAA 3’

Expected wild type signal is C

Expected mutant type signal is G

c.2118_2119delTG

2087 CAAAAATATTAACCATTGACATGGTAATCACTGTTAAGAGACACCCTCAGAAAGTTCACAATCAA 2151

AATTCTCTGTGGGAGTCTTTCAAGTG

Row 1. DNA sample has normal genotype for posit. c. 2118_2119. We have detected in sense strand normal G allele and in antisense strand we have detected normal C allele (G/C).

Row 2. DNA sample is heterozygous for c.2118_2119delTG. We have detected in sense strand normal G allele and mutant T allele. In antisense strand we have detected normal G allele and mutant C allele (GT/CG).