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.
BACKGROUND
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.


METHODS
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 degrees 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.


RESULTS
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 approximately 6.5 kilobases (kb) in length, 2 amplicons approximately 10 kb in length, and 2 amplicons approximately 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.


CONCLUSIONS
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.
Rodriguez
Joel A
JA
Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
Guiteau
Jacfranz J
JJ
Nazareth
Lynne
L
Reid
Jeff G
JG
Goss
John A
JA
Gibbs
Richard A
RA
Gingras
Marie-Claude
MC
D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D005091:Exons / Q000235:genetics; D015723:Gene Library; D016147:Genes, Tumor Suppressor; D006801:Humans; D047668:Inteins / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*
D051059:PTEN Phosphohydrolase