Table 1. PCR primers and conditions for... | American Society of Hematology

Table 1.

PCR primers and conditions for amplification of ALAS2 from genomic DNA

Amplified region	Primer coordinates^*	Primer no.	Temperature, °C	Amplicon, bp	Oligonucleotides†
Proximal promoter	UP17888	768	55	367	5′-GCCGCCAAGCTT-TTGAGGAGATCTATAGTCAGAGAGG-3′
-293 to +28	LP18179	769	55	367	5′-GCCGCCCCATGGTGGCTTTACCAACAGTACCGGAATGCCGAAC-GAATGACAGGTGGGTACTTGG-3′
Mismatch PCR	UP17956	432	60	327	5′-TCCAAAGCCCAAATGAGCTAATCTT-3′
promoter mutation	LP18252	128	60	327	5′-GCCGCCGAATT-CAGCTGGCAGACCAGAGATA-3′
Primer extension	LP18203	2060	71	NA	5′-CTGTTGCCCTGCACTGAGGACG-3′

Amplified region	Primer coordinates^*	Primer no.	Temperature, °C	Amplicon, bp	Oligonucleotides†
Proximal promoter	UP17888	768	55	367	5′-GCCGCCAAGCTT-TTGAGGAGATCTATAGTCAGAGAGG-3′
-293 to +28	LP18179	769	55	367	5′-GCCGCCCCATGGTGGCTTTACCAACAGTACCGGAATGCCGAAC-GAATGACAGGTGGGTACTTGG-3′
Mismatch PCR	UP17956	432	60	327	5′-TCCAAAGCCCAAATGAGCTAATCTT-3′
promoter mutation	LP18252	128	60	327	5′-GCCGCCGAATT-CAGCTGGCAGACCAGAGATA-3′
Primer extension	LP18203	2060	71	NA	5′-CTGTTGCCCTGCACTGAGGACG-3′

UP indicates upper-strand primer; LP, lower-strand primer; and NA, not applicable

Coordinates corresponding to the 5′ ends of the primers are taken from the GenBank genomic clone Z83821

†

The hyphen internal to the sequence indicates the beginning of the ALAS2 genomic sequence. The annealing temperatures are for the regions to the right of the vertical bar. HindIII, NcoI, and EcoRI sites introduced for cloning purposes are underlined in primers no. 768, 769, and 128, respectively. The mismatched cytosine in primer no. 432 is indicated in bold

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