Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue.
Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.
Warford
A
A
Department of Pathology, University of Leicester, U.K.
Pringle
J H
JH
Hay
J
J
Henderson
S D
SD
Lauder
I
I
D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D004586:Electrophoresis; D005404:Fixatives; D005557:Formaldehyde; D006652:Histological Techniques; D006801:Humans; D008214:Lymphocytes / Q000032:analysis; D009693:Nucleic Acid Hybridization; D014066:Palatine Tonsil / Q000032:analysis; D013997:Time Factors
D005404:Fixatives; D005557:Formaldehyde; D004247:DNA; D004262:DNA Restriction Enzymes