Simple quantitative PCR analysis for allelic Pten loss in tumor progression

• Published in: Analytical biochemistry Vol. 526; pp. 50 - 57
• Main Authors: Wu, Xingping, Zhu, Ailin, Pang, Xiaowu, Xie, Guiqin, Gu, Xinbin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2017
• Subjects: Allele-specific copy number; Allele-specific PCR; Alleles; Animals; Disease Progression; DNA Copy Number Variations - genetics; DNA, Neoplasm - genetics; Gene Silencing; Mice; Mutation - genetics; Polymerase Chain Reaction - methods; PTEN Phosphohydrolase - deficiency; PTEN Phosphohydrolase - genetics; Thyroid Gland - metabolism; Thyroid Neoplasms - genetics; Thyroid Neoplasms - pathology; Allele-specific copy number; Allele-specific PCR
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2017.03.016

We describe a simple method to accurately detect and quantify both Pten mutation and allele-specific loss using allele-specific PCR analysis. Our approach used a heterozygous genomic DNA with one wild-type and one mutant Pten allele as a reference at a single concentration to calculate the percent ratio of the wild-type Pten gene for the detection of allele-specific gene loss. With a standard curve, ratios from PCR data were used to quantitate the wild-type Pten allele copy number loss in tumor specimens. We demonstrate the utility of our approach to calculate allele-specific Pten loss during tumor progression and show that our approach generates quantitative data that are comparable to those obtained from digital droplet PCR. As a method to detect both mutation and allele-specific gene loss, our approach is less subject to the variability of sample amount that are often very limited in clinical analysis. Since conventional PCR is easy to be carried out, our method simplifies the workflow in any laboratory and would provide significant advantages for simplicity to quantify allele-specific gene loss. •We established a simple and sensitive allele-specific PCR analysis using a heterozygous genomic DNA as reference.•This method was as accurate as droplet digital PCR to detect allele-specific Pten loss, even with limited genetic materials.•Allele-specific PCR analysis was capable to detect progressive allelic loss during tumor progression.