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)

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

doi:10.1186/s12881-016-0334-y

Table 1 Primers for the HRM-PCR/GRACE-PCR assay of the HBB gene

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)

Primer set	Direction	Primer/Probe sequence	Position on ref sequence	Product length (bp)	Target	Annealing temp (°C)	HRM melt range (°C)
H1	Forward	5’-GGCTGTCATCACTTAGACCTCA-3’	5,227,065 to 5,227,196 on NC_000011.10	132	HBB Promoter	59	80–90
H1	Reverse	5’-CAAATGTAAGCAATAGATGGCTC-3’	5,227,065 to 5,227,196 on NC_000011.10	132	HBB Promoter	59	80–90
H2	Forward	5’-ATTTGCTTCTGACACAACTG-3’	5,226,952 to 5,227,069 on NC_000011.10	118	HBB Exon 1	55	70–90
	Reverse	5’-CTTCATCCACGTTCACCTTG-3’
	Probe^a	5’-CTCCTGAGGAGAAGTCT-GCCGTTACTGCCCTGTGGGG-3’
H3	Forward	5’-TGCCGTTACTGCCCTGT-3’	5,226,886 to 5,226,992 on NC_000011.10	107	HBB Exon 1	59	75–85
H3	Reverse	5’-TTCTATTGGTCTCCTTAAACCTGT-3’	5,226,886 to 5,226,992 on NC_000011.10	107	HBB Exon 1	59	75–85
H4	Forward	5’-CACTGACTCTCTCTGCCTA-3’	5,226,726 to 5,226,841 on NC_000011.10	116	HBB Exon 2	59	80–90
H4	Reverse	5’-TAACAGCATCAGGAGTGGACA-3’	5,226,726 to 5,226,841 on NC_000011.10	116	HBB Exon 2	59	80–90
H5	Forward	5’-GTCTACCCTTGGACCCAG-3’	5,226,674 to 5,226,789 on NC_000011.10	116	HBB Exon 2	59	80–90
H5	Reverse	5’-CTAAAGGCACCGAGCACT-3’	5,226,674 to 5,226,789 on NC_000011.10	116	HBB Exon 2	59	80–90
H6	Forward	5’-GCTCATGGCAAGAAAGTGCTC-3’	5,226,549 to 5,226,705 on NC_000011.10	157	HBB Exon 2	59	80-90
H6	Reverse^b	5’-GAAAACATCAAGIGTCCCA-3’	5,226,549 to 5,226,705 on NC_000011.10	157	HBB Exon 2	59	80-90
H7	Forward	5’-TGCCTCTTTGCACCATTCTA-3’	5,225,893 to 5,225,974 0n NC_000011.10	82	HBB IVS2	55	70–80
H7	Reverse^b	5’-GAAATATTTATATGCAGAIATATTGCTA-3’	5,225,893 to 5,225,974 0n NC_000011.10	82	HBB IVS2	55	70–80
H8	Forward	5’-CTAATAGCAGCTACAATCCAG-3’	5,225,702 to 5,225,852 on NC_000011.10	152	HBB IVS2	56	80–90
H8	Reverse	5’-CACAGACCAGCACGTT-3’	5,225,702 to 5,225,852 on NC_000011.10	152	HBB IVS2	56	80–90
H9	Forward	5’-TTGCTAATCATGTTCATACCTC-3’	5,225,632 to 5,225,765 on NC_000011.10	134	HBB Exon 3	59	80–90
H9	Reverse	5’-CCAGCCACCACTTTCTGAT-3’	5,225,632 to 5,225,765 on NC_000011.10	134	HBB Exon 3	59	80–90
H10/G2	Forward	5’-GAATTCACCCCACCAGTGC-3’	5,225,559 to 5,225,678 on NC_000011.10	120	HBB Exon 3	55	80–90
H10/G2	Reverse	5’-AGGAACCTTTAATAGAAATTGGAC-3’	5,225,559 to 5,225,678 on NC_000011.10	120	HBB Exon 3	55	80–90
H11	Forward	5’-CCCACAAGTATCACTAAGCTC-3’	5,225,423 to 5,225,614 on NC_000011.10	192	HBB Exon 3	55	78–88
H11	Reverse	5’-CCCTTTTTAGTAAAATATTCAGA-3’	5,225,423 to 5,225,614 on NC_000011.10	192	HBB Exon 3	55	78–88
G1	Forward	5’-TGAAGTCCAACTCCTAAGCC-3’	5,227,086 to 5,227,243 on NC_000011.10	158	HBB Promoter	55	75–90
G1	Reverse	5’-TCTGCCCTGACTTTTATGCC-3’	5,227,086 to 5,227,243 on NC_000011.10	158	HBB Promoter	55	75–90
G3	Forward	5’-CACCCGGCCTCATGGAT-3’	1,462,101 to 1,462,255 on NC_000016.10	155	CLCN7 control for GRACE PCR	55	NA
G3	Reverse	5’-AAGAGAACTACAGACCAACACCC-3’	1,462,101 to 1,462,255 on NC_000016.10	155	CLCN7 control for GRACE PCR	55	NA

Primer sets H1 to H11 were used to scan the HBB gene for mutations by HRM. Primers sets G1 and G2 were used to detect deletions of the HBB gene promoter and third exon by GRACE-PCR. Primer set G3 is the internal control for the GRACE-PCR reactions
^aThe probe used in conjunction with primer set H2 is an unlabelled oligonucleotide that has a phosphate block at the 3’ end
^bThe reverse primers of primer sets H6 and H7 contain the universal base inosine, indicated with an I in the primer sequence

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ISSN: 1471-2350

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