An Improved Method for DNA Extraction from Paraffin Section Yan-gao Man, Farid Moinfar, Gary L. Bratthauer, Elizabeth A. Kuhls, Fattaneh A. Tavassoli Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology （ AFIP ） and American Registry of Pathology （ ARP ）, Washington, DC , USA

Introduction Technical issues of DNA extraction ： ◎ Evaluation of deparaffinization ◎ Satisfied hematoxylin stain ◎ Ratio of cell number to enzyme volume ◎ Monitor digestion process

Introduction ◎ Paraffin （ mp ℃ ） ◎ Degree of freshness of xylene ◎ Temperature ◎ Duration ◎ Thickness of section Deparaffinization ：

Introduction Hematoxylin stain ：

Introduction

Introduction ◎ Stable in dissection buffer ◎ Non-lesion on re-cuts ◎ Tissue blocks not accessible ◎ Reducing in 2 ％ hydrochloric acid ◎ Satisfied both morphological and molecular assessments

Introduction Ratio of cell number to enzyme volume ： ◎ Larger number of cells  some cell undigested ◎ Larger enzyme volume  low DNA content per unit

Introduction ◎ No practical mean to monitor enzymatic digestion process Monitor digestion process ：

Materials and Method ◎ US 、 Japan 、 China 、 Austria 、 Italy 、 France ◎ Age of the paraffin blocks ： 3 months to over 30 years Slide prepare ： NameManufactoryNote Microscope slides CMS Slide holders CMS

Materials and Method ◎ Thickness ： 5-7 μ m （ a few at μ m ） ◎ Clean cutting method ： distilled water 、 gloves 、 new blade ◎ Place vertically in 80 ℃ for minutes ◎ Cool down 5-10 minutes at room temp. Slide prepare ：

Materials and Method NameManufactoryNote Xylene Fisher Scientific Ethanol Deparaffinization ： ◎ Xylene 3-5 minutes 3 times ◎ Descending concentration of ethanol 3-5 minutes ◎ Running tap water 5 minutes

Materials and Method ◎ Hematoxylin seconds ◎ Rinse in tap water Stain ： NameManufactoryNote Hematoxylin Fisher Scientific Hydrochloric acid Fisher Scientific Ammonium hydroxide LabChem Inc GlycerinCMS

Materials and Method ◎ Dip in 2 ％ hydrochloric acid 3-5 times ◎ Running tap water 5-7 minutes ◎ 1X PBS （ pH7.4 ） contain 1 ％ ammonium hydroxide 5-7 minutes Stain ：

Materials and Method ◎ Evaluate the extent of deparaffin ：  water retention  hematoxylin stain ◎ Assess hematoxylin stain ：  nucleus  cytoplasm ◎ Distilled water 2-3 minutes ◎ 1X PBS contain 10 ％ glycerin Stain ：

Materials and Method ◎ Deparaffin ◎ Satisfactory stain for morphology ◎ Microdissection ◎ 2 ％ hydrochloric acid 3-5 minutes 2 times ◎ 1X PBS （ pH7.0 ） 5-7 minutes ◎ Centrifuge at g 3 minutes ◎ Digestion A Different Approach ：

Materials and Method NameManufactoryNote Micrometer Olympus Optical 30-Gauge needle Fisher Scientific Mineral oil Fisher Scientific Microcentrifuge tube MJ Research Proteinase K Sigma 10mg/ml ； - 80 ℃ Microdissection & enzymatic digestion ：

Materials and Method ◎ Microdissection ◎ Digestion buffer ：（ fresh made ）  150 μ l proteinase K μl mixture  0.5 ml Tween ml 0.5 M EDTA （ pH8.0 ） ml 1 M Tris （ pH8.5 ） ml distilled water Microdissection & enzymatic digestion ：

Materials and Method ◎ Add digestion buffer  accord number of cells ◎ Mineral oil  equal volume of digestion buffer ◎ ℃ hours （ magnifier ） ◎ Incubate at 95 ℃ 10 minutes ◎ Store at 4 ℃ Microdissection & enzymatic digestion ：

Materials and Method Loss of Heterozygosity ： NameManufactoryNote LOH marker Research Genetics Gene Amp PCR kit Perkin-Elmer Taq gold DNA polymerase Perkin-Elmer Gel loading buffer Perkin-Elmer DNA size standard Perkin-Elmer Polyacryamide gel Bio-Rad

Materials and Method ◎ 30 fluorescent dye-labeled polymorphic DNA markers at 1 、 2 、 3 、 11 、 13 、 16 and 17 ◎ Annealing temperature ： ℃ ◎ 78 to 290 bp ◎ Thermal cycler ： Perkin-Elmer ◎ cycles Loss of Heterozygosity ：

Materials and Method ◎ 5 – 6 ％ Polyacryamide Gel ◎ Gel image ： Perkin-Elmer 377 DNA sequencer ◎ LOH ： 75 ％ reduction of one allele Loss of Heterozygosity ：

Materials and Method ◎ DNA markers on exon 1 of the human androgen receptor gene ◎ Hpa Ⅱ ： methylation sensitive enzyme ◎ Rsa Ⅰ： control enzyme ◎ Monoclonality ： One DNA band or peak after Hpa Ⅱ digestion Clonality analysis ： NameManufactoryNote Hpa Ⅱ Gibco/BRL Life Rsa Ⅰ Gibco/BRL Life

Results and Discussion ◎ 12 μ m thick human breast No pretreate 80 ℃ minutes 1A 放大 1C 放大

Results and Discussion ◎ 12 μ m thick human breast No pretreate 80 ℃ minutes

Results and Discussion No pretreate 80 ℃ minutes D16S518 D3S1300 D11S1311  bp TPO  bp

Results and Discussion 2 ％ hydrochloric acid 1 ％ ammonium hydroxide

Results and Discussion ◎ 10 μl digestion buffer ： cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution （ 10mg/ml ）  re-deparaffinization

Results and Discussion

◎ Incubation of sections at 80 ℃ for minutes ◎ 2 ％ hydrochloric acid & 1 ％ ammonium hydroxide ◎ 10 μl digestion buffer ： cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution （ 10mg/ml ）  re-deparaffinization

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