DISEÑO EXPERIMENTAL

4.1 DNA preparation

DN A w as extracted from the tum our and blood using m odifications of existing protocols.

Extraction of DNA from tumours

This protocol is based on Goeltz et al (1985) and M üllenbach et al. (1989)

Materials

Tris-EDTA, pH 9 (TE9)

500mM Tris base

(60.6g Tris base to IL w ith H2O, adjust to pH 9 using

~ 30ml concentrated HCl) 20mM EDTA

lOmM NaCl 20% Sodium decadoyl sulphate (SDS)

200g SDS in 900ml H2O,

adjust to p H 7.2 by adding a few drops of concentrated HCl, ddH20 to IL.

Proteinase K (BRL, M aryland)

A dd 5ml H2O to lOOmg for stock of 20m g /m l

6M (saturated) NaCl

350.6g NaCl m ade up to IL w ith ddH20. Tris-EDTA (pH 7.4)

lOmM Tris acid pH 7.4 Im M EDTA pH 8 Propan-2-ol

E th an o l

Single-sided razor blade

Plastic Petri dish

Solid CO2 (Cardice)

M e t h o d

1. W eigh a chip of tum our, defrost in a petri dish and slice crudely w ith razor blade.

2. Place petri dish on cardice and allow to re-freeze slightly (to m ake m incing easier) and mince further. Repeat until tu m o u r is semi-solid. 3. Place into 50ml polypropylene tube containing 10ml of TE9, 1% SDS and 500|ig/m l proteinase K.

4. Vortex for 5 seconds to distribute sam ple evenly in buffer 5. Incubate at 48°c for 24 hours.

6. Review sam ple for completeness of digestion. If incom plete, ad d SDS and proteinase K to final concentrations of 2% and I m g /m l

respectively. Incubate for further 12-24 hours.

7. A dd prew arm ed 6M (saturated) NaCl to a final concentration of 1.5M and mix gently (for 10ml add 3.3ml 6M NaCl).

8. A dd an equal volum e of chloroform and mix by gentle rotation for betw een 30 m inutes and 1 hour.

9. Centrifugate at 2000 rpm for 10 minutes.

10. Transfer supernatant w ith an agar pipette into a fresh polypropylene tube and add an equal volum e of propan-2-ol and mix gently to

precipitate the DNA. This can then be recovered by using a cooled, flam ed Pasteur pipette or, if there is little visible DNA, by

centrifugation at 4000 rpm for 20 minutes.

11. W ash the DNA in 70% ethanol for betw een 1 h o u r and overnight. 12. Centrifugate at approx. 2000 rpm for 10 m inutes to pellet the DNA and aspirate off m ost of the ethanol using a 20|il Gilson pipette.

13. Vacuum dry for a few m inutes to rem ove the rem aining ethanol.

14. Resuspend in 500|xl-2ml TE. Leave for a few days at 4°c before assessing DNA quantity and quality.

Extraction of DNA from blood

This protocol is largely th at of M üllenbach et al. (1989) w ith som e

m odifications.

Materials As above: 20% w / v SDS Proteinase K 6M (saturated) NaCl Tris-EDTA (pH 7.4) Propan-2-ol E th an o l In addition: Phosphate-buffered saline 2X Sucrose-Triton: 218.5g sucrose 20mM Tris p H 7.4 (121.1g Tris base to IL w ith d d H2Ü,

adjust to p H 7.4 by adding ~70ml concentrated HCl) 10ml IM M gC b (203.3g MgCl2*6H20 to IL w ith ddH20) 20ml Triton X-100 Make u p to IL w ith H2O Lysis buffer: 75mM NaCl 25mM EDTA( p H 8.0) 1% w / v SDS

For the agarose gel: 100ml 1 X TAE 0.8-2% agarose

Stock 50 X TAE: 484g Tris base, 114.2 ml glacial acetic acid,

200ml 0.5M EDTA, m ade u p to 2L w ith ddH20. M e t h o d

1. C entrifugate non-clotted blood at 1000 rpm for 5 m inutes. 2. Take off plasm a and freeze dow n an aliquot if required.

3. A dd 45ml of 2X Sucrose Triton to w hite cell pellet, place on ice for 10 m inutes.

4. Centrifugate at 4200 rpm for 20 m inutes at 4°c. Repeat steps 3&4 if necessary.

5. Pour off supernatant, taking care not to dislodge pinkish-red pellet 6. Pellet nucleated cells and w ash in PBS to rem ove excess debris. 7. Resuspend in 5-lOml lysis buffer in a 50ml polypropylene tube. 8. A dd proteinase K to a final conc. of ~ 500|ig/m l.

9. Incubate at 48-55°c for 3 hours to overnight. 10. Proceed from step 7 in tum our DNA protocol.

The quality and quantity of DNA obtained was assessed by loading lp.1 of the DNA onto a 0.8% agarose gel, (buffer: 1 X TAE) to g eth er w ith an ap p ro p riate size m arker. The am ount of DNA present and its integrity could be visualised after ethidium brom ide staining and view ing u n d er UV illum ination. In ad d itio n , the optical d ensity w as m easu red by absorption at w avelengths of 260nm (DNA) and 280nm (protein) using a LKB U ltrospec II Spectrophotom eter (Pharm acia). The ratio 260/280 should be 1.6 for DNA that is largely free of protein contam ination.

4.2 Restriction endonuclease digestions

Materials

R e stric tio n e n d o n u c le a se s an d a p p ro p ria te b u ffe rs (B o eh rin g er M annheim UK, Lewes, Sussex, N ew E ngland Biolabs UK, B ishop's Stortford, Herts)

ddPUO

E thanol

3M sodium acetate pH 5.2 (408.Ig sodium acetate* 3H2O to IL w ith H2O

after adjusting to pH 5.2 w ith glacial acetic acid). 200pm sperm idine

M e t h o d

1. A dd DNA (5-lOpg/ml) to labelled tube.

2. Make up reaction mix of lOx restriction endonuclease buffer, 50-100u

of restriction endonuclease and d d H2 0 to desired volum e.

3. Finger vortex briefly, microcentrifugate for 1 second.

4. Incubate at tem perature indicated by m anufacturer for 1 h o u r to o v ern ig h t.

5. Assess digestion on agarose gel of appropriate concentration.

If the DNA fails to completely digest

1. A dd another 50-100u endonuclease, b u t do not exceed 10% final concentration, as glycerol m ay inhibit the reaction.

2. Repeat digestion, adding 200mM sperm idine to a final concentration of 2mM.

3. Purify DNA w ith small volumes of phenol-chloroform follow ed by a final extraction w ith chloroform: isoamylalcohol (in p ro p o rtio n 24:1). Precipitate DNA w ith 1/10 volume 3M sodium acetate an d 2 volum es

100% ethanol, w ash in 70% ethanol and resuspend in 500|xl of TE. Repeat digestion.

Restriction endonuclease digestion of PCR products

In som e instances, digesting PCR pro d u cts w as required. Large SSCP fragm ents can som etim es be digested before loading onto gels, and as show n in C hapter 3, digestion of PCR products am plified using prim ers w ith in in tro n s and exons of A PC can obviate the need for sequence co n firm atio n .

Materials and Method

As above, except the PCR product is used directly. There is no need to purify the product.

In addition:

1. If a 50pl PCR volume is used, check reaction results by loading l-2|xl onto agarose gel.

2. Digest 20-25|xl in 30|il final volume, using appropriate restriction enzym e (5-20u) and buffer for 1 hour.

3. Load onto 1-2% agarose gel w ith undigested DNA as control.