Additional methods information with “Oestrogen receptor α gene haplotype and postmenopausal breast cancer risk: a case control study”

Genetic analyses

Minisequencing assay using fluorescence polarization detection

The region containing the intron 1 single nucleotide polymorphisms (SNPs), c.454-397C>T and c.454-351A>G, was amplified in one PCR fragment. PCR was performed using 20 ng of genomic DNA, 0.02U/µl Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 100 M dNTPs (Amersham Pharmacia Biotech, Uppsala, Sweden) and 0.1 M of both PCR primers in 30 µl of GeneAmp PCR buffer supplied with the enzyme at 95C for 7 minutes, followed by 40 cycles at 92C for 15 seconds, 56C for 1 minute and 72C for 45 seconds. Five microliters of PCR product was aliquoted to 384-well microtiter plates and 5 l of a PCR cleanup mixture containing 0.1 U/µl shrimp alkaline phosphatase (USB Corporation, Cleveland, OH) and 0.1 U/µl Exonuclease I (USB Corporation) in 20mM Tris-HCl pH 8.0 and 10mM MgCl2 was added. The plate was incubated at 37°C for 1 hour, followed by 15 minutes at 95°C to inactivate the enzymes. Five microliters of extension mixture containing 5 µM extension primers, and 0.08 U/µl Thermosequenase (Amersham Pharmacia Biotech) in 50 mM Tris-HCl pH 9, 50 µM KCl, 5 mM NaCl, 5 mM MgCl2, 8% glycerol, and fluorescently labeled and unlabeled ddNTPs was added to each reaction. The two ddNTPs relevant for the particular SNP were labeled and included at a 1:5 ratio relative to unlabeled ddNTPs. The total concentration of ddNTP was 0.125 µM. The fluorescent dye rhodamine 110 (R110) was used to label ddGTP and ddCTP, and 6-carboxytetramethylrhodamine (TAMRA) were used to label ddATP and ddUTP (PerkinElmer Life Sciences, Boston, MA, USA). The cyclic extension reaction was performed at 92°C for 1 minute followed by 40 cycles of 92°C for 10 seconds and 55° for 30 seconds. The fluorescence signals in the 384 well plates were read on an Analyst ADTM (Molecular Devices Corporation, Sunnyvale, CA, USA). Genotypes were assigned using the software AlleleCaller TM supplied with the instrument, and a custom-made Excel macro.

Molecular Beacon assay

To analyze the exonic SNPs at c.975C>G and c.729C>T, 10 ng of genomic DNA was amplified in a 25 l reaction containing 0.02 U/µl of AmpliTaq Gold DNA polymerase (Applied Biosystems), 3.5 mM MgCl2, 250 M of dNTPs (Amersham Pharmacia Biotech) and 1 M of PCR primers in TaqMan Buffer A containing the ROX dye (Applied Biosystems). The two MB probes were included at 0.34 M concentration. The cycling conditions were 95 C for 10 minutes, followed by 40 cycles at 95 C for 30 seconds, 55 C (codon 325) or 57 oC (c.729C>T) for 1 minute and 72 C for 45 seconds and was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems). The increase in fluorescent signal was registered during the annealing step of the reaction and the end-point signals were used to assign the genotypes as previously described (Tapp, I., L. Malmberg, et al. (2000). "Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes." Biotechniques28(4): 732-8).

Solid-phase minisequencing assay

Solid-phase minisequencing in microtiter plates (Syvanen, A. C., A. Sajantila, et al. (1993). "Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing." Am J Hum Genet52(1): 46-59) was used in part to genotype the c.729C>T SNP.

Microsatellite assay

The TA-repeat region was amplified using an ABI-877 Integrated Thermal cycler PCR robot with standard reagents (Applied Biosystems) (Holgersson, S., J. A. Karlsson, et al. (1994). "Fluorescent-based typing of the two short tandem repeat loci HUMTH01 and HUMACTBP2: reproducibility of size measurements and genetic variation in the Swedish population." Electrophoresis15(7): 890-5). The cycling conditions were 96°C for 10 minutes followed by 36 cycles of 30 seconds at 96° C, 30 seconds at 57° C, and 1 minute at 72° C with a final extension step for 7 minutes. The HEX-labeled fluorescent PCR products were mixed with an internal-lane standard (GS-500 TAMRA) and separated on a 6% polyacrylamide gel using a 96-well ABI 377 automatic sequenator. Gel data were analyzed using Genescan Analysis 3.1 and allele sizes were determined using Genotyper 2.0 (all Applied Biosystems).

Additional methods information with “Oestrogen receptor α gene haplotype and postmenopausal breast cancer risk: a case control study”

Table 1

PCR primers used to genotype estrogen receptor alpha
Sequence 5’ -> 3’ and modifications
Polymorphism / Forward / Reverse
Promotor TAn a / HEX - GAT TAT AGA CGC ATG ATA TAC TTC ACC / GGA TAT GCA GAA TCA AAT ATC CAG ATG
Intron 1 c.454-397C>T and c.454-397A>G b / (Bio -) GTG TTG TCC ATC AGT TCA TCT / (Bio -) AGA ACC ATT AGA GAC CAA TGC
Exon 3 c.729C>T (MB) c / CCA ACC AGT GCA CCA TTG ATA / CCT TTC ATC ATT CCC ACT TCG
Exon 3 c.729C>T d / AAC AGG AGG AAG AGC TGC CAG GC / Bio - CTT TCA TCA TTC CCA CTT CGT AGC A
Exon 4 c.975C>G (MB) c / CAG ATG GTC AGT GCC TTG TTG GA / CGA AGC TTC ACT GAA GGG TCT GG
Exon 4 c.975C>G d / TGA CGG CCG ACC AGA TGG TCA GT / Bio - AGG GTC TGG TAG GAT CAT ACT CGG

a Forward primer labeled with 6-carboxyhexachlorofluorescein (HEX) to enable detection on the sequencing instrument.

b Primers used for minisequencing with fluorescence polarization detection. For validation by solid-phase minisequencing one of the PCR primers was substituted by a biotinylated (Bio) primer with the same sequence to enable solid-phase capture on streptavidin coated microtiter plates.

c Primers optimized for the molecular beacon assay (MB)

d For validation by solid-phase minisequencing we used a second pair of PCR primers, of which the reverse primer was biotinylated (Bio) to enable solid-phase capture on streptavidin coated microtiter plates.

Table 2

Primers and probes used for genotyping the estrogen receptor alpha gene
Assay / Polymorphism / Sequence 5’ -> 3’ and modification
Minisequencing / Intron 1 c.454-397C>T a / Non-coding / TGG GAA ACA GAG ACA AAG CAT AAA AC
Coding / CAT CTG AGT TCC AAA TGT CCC AGC
Minisequencing / Intron 1 c.454-351A>G a / Non-coding / GAC CAA TGC TCA TCC CAA CTC
Coding / TCC CAG AGA CCC TGA GTG TGG TCT
Molecular Beacon / Exon 3 c.729C>T b / FAM - ccg agc CGG CTC CGC AAA TGC TAg ctc gg - DABCYL
TET - ccg agc CGG CTC CGT AAA TGC TAg ctc gg - DABCYL
Molecular Beacon / Exon 4 c.975C>G b / FAM - cca agc GAG CCC CCC ATA CTC TAg ctt gg - DABCYL
TET - cca agc GAG CCC CCG ATA CTC TAg ctt gg – DABCYL
Minisequencing / Exon 3 c.729C>T c / TGC CAG GCC TGC CGG CTC CG
Minisequencing / Exon 4 c.975C>G c / TTG TTG GAT GCT GAG CCC CC

a Primers used for minisequencing with fluorescence polarization detection in both directions and for validation by solid-phase minisequencing.

b Two molecular beacon probes, each corresponding to one of the variable nucleotides and labeled with 5-carboxyfluorescein (FAM) or 6-carboxytetrachlorofluorescein (TET) and dimethylaminophenylazobenzoic acid (DABCYL) were used in the same reaction in the molecular beacon assay. Small letters indicate the stem part and bold letters indicate the nucleotide complementary to the SNP position

c Primers used for validation by solid-phase minisequencing.