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Table 2 Primers used to generate amplicons for sequencing and Gap-PCR

From: Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR)

Target

Direction

Primer sequence

Position on reference sequence

Product length (bp)

Annealing temp °C

Extension time (sec)

HBB Exon 1 and 2

Forward

5’-TGTCATCACTTAGACCTCACCCTG-3’

5,226,544 to 5,227,193 on NC_0000.11.10

686

58

40

Reverse

5’-GGAAAGAAAACATCAAGCGTCCCATAG-3’

HBB Intervening sequence 2

Forward

5’-TGCACGTGGATCCTGAGAACTTCA-3’

5,226,411 to 5,226,602 on NC_000011.10

228

56

20

Reverse

5’-ACAGCAAATAAAAGAAACTAAAACGA-3’

HBB Exon 3

Forward

5’-GCTGGATTATTCTGAGTCCAAGCTA-3’

5,225,508 to 5,225,797 on NC_000011.10

326

58

25

Reverse

5’-TCAAGGCCCTTCATAATATCCCC-3’

GAP-PCR Filipino β-thalassaemia deletion

Forward

5’-GTAAATGAGTAAATGAAGGAATGAT-3’

5,112,653 to 5,232,062 on NC_000011.10

920

60

60

Reverse

5’-TGTGATTTGGCTCTCTTCTTGTCTA-3’

  1. The primers used to generate amplicons for sequencing the HBB gene are shown along with the appropriate PCR reaction conditions. The sequencing primers were synthesized with M13 tails. Primers used for the detection of the Filipino β-thalassaemia deletion by Gap-PCR are also shown (without M13 tails)