Genomic DNA extraction methods using formalin-fixed paraffin-embedded tissue

Autor: Michael N. Kent, Keerti Potluri, Ahmed Mahas, Michael P. Markey, Sameep Naik
Rok vydání: 2015
Předmět:
Zdroj: Analytical Biochemistry. 486:17-23
ISSN: 0003-2697
DOI: 10.1016/j.ab.2015.06.029
Popis: As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues.
Databáze: OpenAIRE
Externí odkaz: