Breast Cancer Research

Table 1 PCR and dHPLC conditions

From: Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR)

						PCR		DHPLC
Gene	Exon	Domain	Forward PCR Primer (5'to 3')	Reverse PCR Primer (5' to 3')	Amplicon Size (bp)	Annealing Temperature (°C)	MgCl (nM)	Melting Temperatures (°C)
SMAD3	7	MH2	CGGCAGTGCCCATTTCCCCTAC	CTAATCCAATCACCTCCAGATT	450	60	3	60, 62.5
SMAD3	8	MH2	TATAAATGAGGCTGGTCTAGGG	GACATGCCTACTACGACCGTAG	544	60	2	60.2, 62.2
SMAD3	9	MH2	GTTTAACTCTTTAAAGTCGACT	ACAGCTGTTCATAACATCCACC	556	60	2	58
SMAD4	8	MH2	TTTAAGAACAGTGCTAAGTACT	TTAAGATGGAGTGCTTACAAAT	566	60	4	51.5, 53.5
SMAD4	9	MH2	TTTAATTTTTCAATATTAAGCA	TAGATTACTGATAATGTCAATA	411	54	4	51.5
SMAD4	10	MH2	TAATGAAACTGAGTTTTAAATAA	ATTTTACCAATTCAAAAATGTCA	377	57	3	51, 53
SMAD4	11	MH2	CTTTAGCAGAGAAGTTATATGCT	AATATATCTTCAGATTATAAACA	424	57	4	59

Polymerase Chain Reaction (PCR) cycle conditions: four minutes 94°C initial denaturation; 94°C for 30 seconds, 0.5 to 1 minute at specified temperature (Ta), 72°C for 0.5 to 1 minute for 35 cycles; and a final extension step of seven minutes at 72°C. Where applicable, Denaturing High-Performance Liquid Chromatography (DHPLC) was run at two melting temperatures to obtain optimal separation.

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ISSN: 1465-542X

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