DNA extraction was performed using a Puregene DNA Purification Kit (Gentra Systems, Big Lake, MN). The PCR thermal cycling conditions, primers used for amplification and genotyping of TP53 G72C Arg/Pro (rs1042522), have been described previously.12 The DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Little Chalfont, United Kingdom) was used to sequence the amplified fragment on the MegaBase 1000 sequencer (Applied Biosystems, Foster City, CA). Results were aligned to the reference sequence using SeqManII sequence analysis software (DNASTAR, Madison, WI) to determine the SNP genotype.
Megabase 1000 sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MegaBase 1000 is a high-throughput DNA sequencer designed for large-scale genomic projects. It utilizes Sanger sequencing technology to generate accurate DNA sequence data. The MegaBase 1000 can process multiple samples simultaneously, providing a streamlined solution for researchers and laboratories requiring efficient DNA sequencing capabilities.
Automatically generated - may contain errors
2 protocols using megabase 1000 sequencer
1
TP53 Genotyping by Sequencing
2
DNA Extraction and SNP Genotyping
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DNA was extracted from cultured fibroblasts using Puregene DNA Purification Kit (Gentra System, Qiagen, Minneapolis, MN, USA) according to the manufacturer's instruction. PCR primers of the selected SNPs are available upon request. Relevant segments of DNA were amplified by thermal cycling as described previously [19] . The amplified fragment was directly sequenced using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instruction, and were run on the MegaBase 1000 sequencer (Applied Biosystems, Waltham, MA, USA). Sequencing results were aligned to the corresponding reference sequence and the SNPs were genotyped using SeqManII sequence analysis software (DNASTAR Inc., Madison, WI, USA).
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