Sequencing the Full-Length of the Phosphatase and Tensin Homolog (PTEN) Gene in Hepatocellular Carcinoma (HCC) Using the 454 GS20 and Illumina GA DNA Sequencing Platforms
- 264 Downloads
Phosphatase and tensin homolog (PTEN) is a tumor-suppressor gene that is mutated in cancer of the liver, pancreas, endometrium, and prostate. PTEN-dependent pathways are involved in mediating cell growth and invasion. To sequence the whole gene (including introns and exons), we have taken advantage of new technologies that allow for rapid, inexpensive sequencing to great depth.
DNA from 15 HCC specimens were pooled, and long-range PCR was performed by using the GeneAmp XL PCR kit. Primer parameters included: length of 20–30 base pairs (bp), melting temperature of −68°C, and G/C content of 50–60%. PCR products were then column-purified and pooled, and DNA libraries were prepared for “shotgun sequencing” on both the 454 GS and Illumina GA sequencing platforms.
We successfully amplified approximately 98.9% of the PTEN gene by using one long-range PCR protocol applied to 24 primer sets, resulting in 20 amplicons ~6.5 kilobases (kb) in length, 2 amplicons ~10 kb in length, and 2 amplicons ~2.5 kb in length. Sequencing of fragmented PCR products on both sequencing platforms identified six high-frequency SNPs that were catalogued in dbSNP as known variants.
Shotgun sequencing based on a single long-range PCR protocol in pooled samples is an efficient and relatively inexpensive way to sequence an entire gene.
The project was supported by grants from the NIH–National Human Genome Research Institute (Grant #2 U54 HG003273) and the Effie and Wofford Cain Foundation. The authors thank the HGSC faculties who contributed to the completion of this work, including Donna M. Muzny, Yi Han, John McPherson, David A. Wheeler, R. Gerald Fowler, and David N. Parker; Huyen H. Dinh, Sandra Lee, Christie L. Kovar, and Michael Holder from the HGSC 454 Group; James C. Durbin, Anthony San Lucas, and Adam M. Dunn from the HGSC Bioinformatics Group; Kaiyi Li for assistance with DNA extraction of banked liver tissue; and Drs. Changyi Chen and F. Charles Brunicardi for their support and advice.