Autogen flexigene

TCF4 TNR length was determined as described previously.^{4 (link),6 (link)} Briefly, genomic DNA was isolated from leukocytes using QIAGEN AutoGen FlexiGene and suspended in TE buffer at a final concentration of 250 ng/μL. TCF4 TNR regions were PCR amplified using 100 ng genomic DNA and 10 pmol oligonucleotide primers 5-TCF-Fuchs and 3-TCF-Fuchs1 in the presence of Invitrogen Platinum PCR Super Mix High Fidelity. PCR program consisted of a single cycle at 95°C for 6 minutes followed by 35 cycles at 95°C for 1 minute, 62°C for 1 minute, and 68°C for 3 minutes. Final 68°C incubation for 7 minutes was performed, at which time the sample was refrigerated at 4°C. Primer sequences for 5-TCF-Fuchs and 3-TCF-Fuchs1 have been described previously.^{4 (link)}For Short Tandem Repeat analysis, a 5′ FAM primer was used in place of 5-TCF-Fuchs and PCR was performed as described above. After PCR amplification, 2 μL DNA was mixed with 12 μL diluted Map Marker 1000 (BioVentures, Inc., Murfreesboro, TN, USA) and TCF4 TNR determination was performed using GeneScan on an ABI 3730XL DNA Analyzer (Foster City, CA, USA). 5′ FAM primer sequence was described previously.^{4 (link)}

TCF4 TNR length was determined as described previously.^{14 (link)} Briefly, leukocyte-derived DNA was extracted using AutoGen FlexiGene (Qiagen) and resuspended in 1× Tris-EDTA (TE) buffer for a final concentration of 250 ng/μL. TNR repeats from each sample were amplified by PCR, using an i-cycler (Bio-Rad, Hercules, CA, USA) by placing 100 ng of genomic DNA with 10 pmol of oligonucleotide primers specific for TCF4 (5-TCF-Fuchs': 5′-CAGATGAGTTTGGTGTAAGATG-3; and 3-TCF-Fuchs' 1: 5′-ACAAGCAGAAAGGGGGCTGCAA-3′) in the presence of Platinum PCR Super Mix High Fidelity (Invitrogen, Carlsbad, CA, USA). The PCR program consisted of 100 ng of genomic DNA and 10 pmol of each primer in 50 μL of Hot Start (Invitrogen) at 95°C for 6 minute for 1 cycle, then 95°C for 1 minute for 1 cycle, then 62°C for 1 minute, followed by 68°C for 3 minute for 35 cycles, and then 68°C for 1 cycle for 7 minute, followed by a 4°C hold.
For short tandem repeat analysis, a 5′FAM primer (5-FAM-TCF-Fuchs': 5′-CAGATGAGTTTGGTGTAAGATG-3′) was used in place of 5-TCF-Fuchs', and PCR was performed as described above. After PCR amplification, 2 μL of DNA was mixed with 12 μL of diluted Map Marker 1000 (Bio Ventures, Inc., Murfreesboro, TN, USA). Gene Scan was carried out using 3730XL DNA analyzer (ABI, Foster City, CA, USA).