1. 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 20306-6000, USA

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

3. Introduction ◎ Paraffin （ mp 40-60 ℃ ） ◎ Degree of freshness of xylene ◎ Temperature ◎ Duration ◎ Thickness of section Deparaffinization ：

4. Introduction Hematoxylin stain ：

5. Introduction

6. 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

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

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

9. 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

10. Materials and Method ◎ Thickness ： 5-7 μ m （ a few at 10- 12 μ m ） ◎ Clean cutting method ： distilled water 、 gloves 、 new blade ◎ Place vertically in 80 ℃ for 30-60 minutes ◎ Cool down 5-10 minutes at room temp. Slide prepare ：

11. 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

12. Materials and Method ◎ Hematoxylin 30-60 seconds ◎ Rinse in tap water Stain ： NameManufactoryNote Hematoxylin Fisher Scientific Hydrochloric acid Fisher Scientific Ammonium hydroxide LabChem Inc GlycerinCMS

13. 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 ：

14. 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 ：

15. 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 2000-3000g 3 minutes ◎ Digestion A Different Approach ：

16. 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 ：

17. Materials and Method ◎ Microdissection ◎ Digestion buffer ：（ fresh made ）  150 μ l proteinase K + 850 μl mixture  0.5 ml Tween 20 + 0.2 ml 0.5 M EDTA （ pH8.0 ） + 5.0 ml 1 M Tris （ pH8.5 ） + 94.3 ml distilled water Microdissection & enzymatic digestion ：

18. Materials and Method ◎ Add digestion buffer  accord number of cells ◎ Mineral oil  equal volume of digestion buffer ◎ 37-40 ℃ 24-48 hours （ magnifier ） ◎ Incubate at 95 ℃ 10 minutes ◎ Store at 4 ℃ Microdissection & enzymatic digestion ：

19. 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

20. Materials and Method ◎ 30 fluorescent dye-labeled polymorphic DNA markers at 1 、 2 、 3 、 11 、 13 、 16 and 17 ◎ Annealing temperature ： 55 - 60 ℃ ◎ 78 to 290 bp ◎ Thermal cycler ： Perkin-Elmer ◎ 30 - 40 cycles Loss of Heterozygosity ：

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

22. 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

23. Results and Discussion ◎ 12 μ m thick human breast No pretreate 80 ℃ 30-60 minutes 1A 放大 1C 放大

24. Results and Discussion ◎ 12 μ m thick human breast No pretreate 80 ℃ 30-60 minutes

25. Results and Discussion No pretreate 80 ℃ 30-60 minutes D16S518 D3S1300 D11S1311  160-300bp TPO  106-130bp

26. Results and Discussion 2 ％ hydrochloric acid 1 ％ ammonium hydroxide

27. Results and Discussion ◎ 10 μl digestion buffer ： 100-150 cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution （ 10mg/ml ）  re-deparaffinization

28. Results and Discussion

30. ◎ Incubation of sections at 80 ℃ for 30-60 minutes ◎ 2 ％ hydrochloric acid & 1 ％ ammonium hydroxide ◎ 10 μl digestion buffer ： 100-150 cells ◎ Monitoring the digestion process with a magnifier ◎ >24 hours digestion  more concentrated enzyme solution （ 10mg/ml ）  re-deparaffinization

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