Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.
Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.
Royal Canadian Mounted Police, National Services and Research, 1200 Vanier Parkway, Ottawa, Ontario K1A 0R2, Canada.
Kristin
K
Duval
Rémy A
RA
Aubin
James
J
Elliott
Ivan
I
Gorn-Hondermann
H Chaim
HC
Birnboim
Derek
D
Jonker
Ron M
RM
Fourney
Chantal J
CJ
Frégeau
D001331:Automation / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000235:genetics; D000431:Ethanol; D005404:Fixatives; D005554:Forensic Medicine / Q000379:methods / Q000639:trends; D005557:Formaldehyde; D005784:Gene Amplification; D056945:Hep G2 Cells; D006801:Humans; D008099:Liver / Q000737:chemistry; D018895:Microsatellite Repeats / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014021:Tissue Preservation / Q000379:methods
D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde; D000431:Ethanol; D004247:DNA