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Rzem et al. 10.1073/pnas.0404840101.

Supporting Information

Files in this Data Supplement:

Supporting Table 1
Supporting Figure 6




Table 1. Primer sequences used for L2HGDH exon amplification (single-stranded conformation polymorphisms, heteroduplex, and sequencing)

 

Forward sequences

Reverse sequences

Length, bp

Exon 1

5'-tcaagtggcttcttctgagc-3'

5'-aggggcagcagcgcagg-3'

231

Exon 2

5'-GCGAGGTCTACTGTAAATATG-3'

5'-CCCAAGTAAGCCCAAAGAAC-3'

207

Exon 3

5'-TTGTATGAATCATGTACTTACTC-3'

5'-AAATGCTAGTTGGAAATTAGTC-3'

245

Exon 4

5'-TACATTCACCTCTATCCATGC-3'

5'-GGACTAAGCCCTAAATAAAAA-3'

219

Exon 5

5'-GCAAGTATTATTTCTCTTATTTG-3'

5'-AGGGCTGACTATATTCAATAG-3'

252

Exon 6

5'-GTAAGGTGCAATCATAGTAATG-3'

5'-GGAAAGAAAGTAGCCAGCAG-3'

164

Exon 7

5'-CCACTTTATACAAATAACTGTTC-3’

5'-CACCTGCTTGAAAAAAATGAG-3'

251

Exon 8

5'-ATGGAATGTGAAATGAGGCTG-3'

5'-ATTGAAAATATAAGCACATAAAATC-3'

250

Exon 9

5'-AAGAATTCCTTTCTCTCATAAAG-3'

5'-ACTGTATTTACACTCCTTATCC-3'

209

Exon 10

5'-TCTTTGAGTCAGCTGACTCC-3'

5'-CATGAAGATTACAGTGCATACC-3'

272

Amplification of the exon 9 deletion

5'-ATGGAATGTGAAATGAGGCTG-3'

5'-TCTAAGGATTCAAACATGCTGG-3'

4,700




Fig. 6. Detection of mutations K81E (family 2) and E176D (family 3) by restriction analysis of PCR amplification fragments. The K81E mutation introduces an additional EarI restriction site in exon 2, and the E176D mutation introduces an additional BamHI site in exon 4. Before restriction, the PCR fragments were denatured for 5 min at 96°C followed by slow renaturation (the temperature was dropped progressively at a rate of 1°C per min until it reached 30°C). The renatured PCR products were then purified by using the Qiagen (Valencia, CA) PCR Purification kit. (Left) DNA samples from subjects homozygous (EE) or heterozygous (KE) for the mutation K81E and from two controls (KK). (Right) DNA samples from subjects homozygous (DD) or heterozygous (ED) for the mutation E176D and from a control (EE).