Table 1 of
Yip, Mol Vis 2007;
13:2183-2193.Table 1 of
Yip, Mol Vis 2007;
13:2183-2193.
Table 1. PCR amplification and denaturing high performance liquid chromatography analysis: primers and conditions
The name of the fragment indicates the exon of the CHM gene amplified (e.g., chm1 for exon 1; two separate fragments chm5.1, and chm5.2 for exon 5). Column temperatures are indicated as astersk if sequence variations were found in the corresponding PCR fragments. Sequence variations were detected in five PCR fragments: chm2, chm5.2, chm6, chm8, and chm13. Optimization of the PCR amplifying fragment chm9 failed probably because of its low GC content (24.5%) and the presence of short tandem repeats within the fragment. (Cycle sequencing of cDNA prepared from a subsequent second sample did not reveal any sequence variation in exon 9 in the samples tested).
DHPLC conditions Fragment PCR primers and conditions ---------------------------- --------- ------------------------------------------------- Column Percent of Name Mg2+(mM)/Tm temperature buffer B (size) Primers (5'-3') (°C) (°C) at start/end --------- ---------------------------------- ------------ ------------- ------------ chm1 F: CTTCAACCCTCCAGGCTAAAT 1.5/58 56, 59 56/65 (360 bp) R: CAAAACTCGCCACTGACAGAA chm2 F: CTCTTTGGTGGTTAAACTGTTCTATACA 2.5/60 52*, 53*, 55/64 (324 bp) R: ATTAAACAACTCCGATCTTTCTTCTAA 55, 57, 58 chm3 F: TAAGGGTTAAAGATGGTTTGTTGATG 1.5/58 53, 54, 55, 52/61 (236 bp) R: TTTCTTCAGTGCAGGGTTACTATGTA 57 chm4 F: GAGTTTTGGTAAGATGAAATAAGCATGA 1.5/58 54, 55, 56, 56/65 (358 bp) R: CAGCATATTAACGTGACTTGTAGCA 57 chm5.1 F: TCATATTTTACTGAAACATTAACAGATGAAT 2.5/58 54, 55, 57 56/65 (382 bp) R: ACAAGTTTTATCATCACAATGGTTTTCT chm5.2 F: TGAAATGCTCACAGAACAAACCC 1.5/58 54*, 57*, 55/64 (324 bp) R: AGAGAATTACAAAAACTGGGTTTATGG 58 chm6 F: TTCTGAGCCTGTAATAGATTGTGTTAT 1.5/58 50, 51, 53*, 56/65 (384 bp) R: GGAATTTACTGCTTTATGAGGAAAATA 54*, 55, 57 chm7 F: CTGTTATATGTAAAGGGGAAAAAAGTG 2.5/60 52, 53, 55, 54/63 (278 bp) R: GAGCAAGCATATTAAAATAGTAAGAAATGT 57 chm8 F: GAAACACACTTCATCTCCTTTTTGT 2.5/58 53, 54, 55*, 57/66 (419 bp) R: TTGCATATACTAAATCTTGAATAAATCTCA 57, 58 chm9* F: CTGTAATACCAGTTATGAAGTGTAAAATA - - (335 bp) R: TTGGGATATGTGTGTGTGTATATGTATA chm10 F: GGTAGCCGATTACATTTTCTCAATTATT 1.5/60 52, 53, 55, 52/61 (246 bp) R: AAGAACAGAATTACACAGGAATGTCA 57, 59 chm11 F: GAGGTGACACTTTTATCCTGACTT 2.5/58 52, 54, 56, 54/63 (279 bp) R: TTAGCTTGAGTGTAGTGATTAGTTCA 57 chm12 F: CAGACATGATGTTTGAAAAAGATACTCA 1.5/60 54, 55, 56, 59/61 (278 bp) R: CTGTAATAACTGCCCCCTTTACC 58, 59 chm13 F: CTTGCTCAGCTCTCTATTATCC 1.5/60 53*, 54, 55 53/62 (262 bp) R: TCTAAGAAGATTATGATGGTTACATTAC chm14 F: TAGACTTCTCTCCTCCCAGA 1.5/58 52, 54, 56, 55/64 (326 bp) R: TCGGTAATAGGCTACACAGTG 57 chm15.1 F: GTACTGTAGCCTGTTTAAGCAAAT 1.5/58 52, 55, 58, 54/63 (304 bp) R: CCTCTAGGTTTCCAAGGTTTG 59 chm15.2 F: GAGGCTAACTCGGAGACTTTC 1.5/58 53, 55, 56, 56/65 (354 bp) R: GATCACTTTCATATCCACAGTTACA 57 |