An improved method for DNA extraction from paraffin sections.
The acquisition of comparable quality and quantity of DNA extracts is the prerequisite to the success of comparative genetic analyses. Although several DNA extracting protocols on paraffin sections have been introduced, the importance of deparaffinization, the procedure for obtaining an adequate hematoxylin staining, the significance of the ratio of the cell number to the enzyme volume, and a practical means for monitoring the digestion process have not been sufficiently addressed. These, however, are the most important factors accountable for a failure of DNA extraction. To minimize the impact of these factors, we have developed several unique strategies, including: (1) incubating sections at 80 degrees C for 30-60 minutes prior to xylene treatment, (2) checking each section to insure the complete removal of paraffin; (3) treating hematoxylin stained sections or cells with de-staining solutions; (4) using a micrometer inserted into the eyepiece of a microscope to estimate the number of cells collected and adjusting the enzyme volume according to the cell number; and (5) monitoring the digestion process with a magnifier. With these strategies, we have been able to consistently obtain comparable quality and quantity of DNA extracts which yielded uniform PCR products regardless of variations in tissue embedding and processing.
Man
Y G
YG
Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology and American Registry of Pathology, Washington, DC 20306-6000, USA. man@afip.osd.mil
Moinfar
F
F
Bratthauer
G L
GL
Kuhls
E A
EA
Tavassoli
F A
FA
D002999:Clone Cells; D004247:DNA / Q000302:isolation & purification*; D004210:Dissection; D005260:Female; D006801:Humans; D019656:Loss of Heterozygosity; D008846:Micromanipulation; D009693:Nucleic Acid Hybridization; D010232:Paraffin / Q000737:chemistry; D016612:Paraffin Embedding; D013194:Staining and Labeling
D010232:Paraffin; D004247:DNA