Aplicativo web

A im : Identifying the parental origin of the cells.

P r i n c ip l e : C ellu lar genom ic DNA w as c u t w ith re s tric tio n endonuclease, sep arated by electophoresis, and hybridized w ith probe. C om parison betw een the p a ttern s of these sm all fragm ents w as perform ed to find evidence of genetic linkage and m utation.

2-8-2 Materials and m ethods

D etailed m ethods and m aterials can be found in the Appendix I. A sum m ary of the whole procedure is presented here.

M & M 56 2-8-2-1 E xtraction of the genomic DNA

A pproxim ately 1.5x10'^ cells were harvested using trypsin or EDTA an d w ashed 3 tim es in PBS. Cells were d ispersed in a 1.8 m is E ppendorf tube containing 1 ml of lysis buffer, 100 pis of 10 m g/m l proteinase K, 250 pis of 10%(m/v) SDS and incubated a t 50^C w ith gentle sh ak in g overnight. DNA w as extracted from lysate by the addition of an equal volum e of phenol(xl), chloroform /phenol(x2), chloroform (xl) an d p recip itated in 2 volum es of ice cold 100% ethanol an d a h alf volum e of 7.5M am m onium acetate. DNA w as rin sed tw ice w ith 70% eth an o l, dried in air, an d dissolved in

lOOpls of TE.

The quality of DNA w as checked by electrophoresis in a 0.8% (w/v) agarose gel w ith TBE as electrophoresis buffer a t 80 volts for 45 m inutes. The quantity of DNA w as m easured by a DNA fluorom eter [TDK 100, Hoefer Scientific In stru m en t, USA] an d ad ju sted to a concentration of 250-500 p g /m l in TE.

2-8-2-2 D igestion of DNA w ith restriction endonuclease

60 pis of sam p le DNA w as c u t by th e ad d itio n of 4 pis of endonuclease H aelll (12u/pl), 8 pis of (lOx)Haelll buffer and 8 pis of 0. l% (w/v) BSA and in cu b ated a t 370C overnight. The digested DNA w as ex tra cted by th e ad d itio n of a n eq u al volum e of ph en o l/ch lo ro fo rm (xl), chloroform (xl) an d purified in ice cold 100% ethanol w ith 2.75M sodium acetate, before dissolving in 20 pis of TE.

2 -S -2 -3 S e p a ra tio n of th e DNA fra g m e n ts bv a g aro se gel electro p h o resis

All the DNA sam ples and a lam bda ladder m ark er w ere ru n in a 0.8% (w/v) agarose gel w ith TBE as electrophoresis buffer a t 24 volts for 48 hours, th en ru n at 36 volts, after ru n n in g buffer w as

M & M 57 changed, for an o th er 24 hours. DNA fragm ents were separated on the agarose gel and d en atu red in situ. The fragm ents were th en transferred from the gel to a solid su p p o rt (m em brane), w here they w ere im m obilized. After preh y b rid izatio n to reduce nonspecific hybridization by the probe, the m em brane w as hybridized to the d esired rad io lab eled nucleic acid probe. The m em b ran e w as w ashed to rem oved u n b o u n d and w eakly binding probe, and w as th en autoradiographed.

2-8-2-4 T ransferring DNA to a m em brane bv S outhern blotting The gel w as im m ersed in 0.25 N HCl for 20 m inutes to depurinate the DNA, in G S l solution for 45 m inutes to d enature the DNA and in GS2 solution for 45 m in u tes to neutralize the gel. The DNA frag m e n ts on th e gel w ere tra n s fe rre d to a n itro c ellu lo se m em brane [Zeta-Probe GT blotting m em brane, Bio-RAD] by the s ta n d a rd S o u th e rn b lo ttin g p ro ced u re w ith (xlO)SSC as the transferring buffer. Briefly a sh eet of nitrocellulose m em brane w as p u t on top of the gel w hich w as placed on a W hatm an 3 MM paper wick tray. Two sh eets of W hatm an 3 MM p ap er w ith a pile of a b so rb an t pap er w ere p u t on top of the m em brane to keep up a stead y capillary p ressu re. D am p a b so rb an t p ap er w as replaced several tim es in th e first h o u r th e n the tra n sfe r pyram id left overnight. M em brane w as w ashed w ith (2x)SSC and baked a t 80°C in a vacuum oven for 30 m inutes.

2 -8 -2 -5 P ro d u ctio n of th e probe and h v b rid izatio n w ith the m em brane

The probe w as a segm ent of ®^P radiolabeled RNA w hich w as synthesized from a specific DNA tem plate in serted down stream from a very specific b acterio p h ag e tran scrip tio n prom oter. The pSPT 18.15 w as u sed as a vector an d linearlized w ith th e

M & M 58 restriction enzym e H indlll. T ranscription reaction w as carried out w ith T7 RNA polym erase and stopped by the addition of nick stop mix. The unincorporated nucleotides were removed by spin colum n c h ro m a to g ra p h y th ro u g h a 1 ml colum n of S ep h ad ex G50 equilibriated w ith TE. The radioactivity of the probe w as m easured w ith a liq u id s c in tilla tio n a n a ly se r. T he m e m b ra n e w as p reh y b rid ized w ith a so lu tio n co n ta in in g 1% BLOTTO (w/v), 2% (w/v) SDS, an d (xl)SSC a t 64°C overnight. 6x10'^ cpm RNA probe w as added to the sam e cham ber and hybridized a t 65^0 overnight. The m em brane w as w ashed in a shaking w ater b a th at 65°C for 45 m in u tes w ith 3 changes of w ash solution containing (xl)SSC, 0.1% (w/v) SDS and w rapped in S aran w rap after it had been air dried.The m em brane w as exposed to X-ray film and kept at -70°C for 2 days w ith two intensifying screens followed by 14 days w ith o u t sc ree n s. Film s w ere developed an d DNA frag m en ts com pared.

2-9 Assay of cellular DNA content by flow cytometry