DOPPCR OF DNA FOR COMPARATIVE GENOMIC HYBRIDIZATION

IndesigningthePCRreaction,besuretoincludeanegativePCRcontrol(blankwithoutDNA),apositivecontrol(weuseMPE-600breasttumorcelllineDNA)andenoughreferenceDNAforalloftheplannedCGHreactions.

METHOD:

1. Add1ul(~50ng)DNAtoeachofthesampletubes.

2. Add5ulofthePre-Amplificationmixtoeachtube.

3. Overlaywith20uloilandstartPCRProgram#91.ThisstepisonlynecessaryifTopo-1preamplificationisused.

4. PCR#91:30min@37C;linkto#92.

5. PCR#92:10min@96C;linkto#93.

6. DuringProgram#92makeupa1:8dilutionofSequenaseinSequenasedilutionbuffer(enoughfor5aliquotsof0.3ulforeachsample).

7. PCR#93:5min@30C,2min@37C,1min@94C,10sec@30C,5cycles.Thenlinkto#94.

8. Forall5cyclesof#93,Sequenase(0.3ul)isaddedthroughtheoilduringtheinitial5mins.

9. Add4.0ulofTaqPolymerase(0.4ulpersample)totheremaining450ulofPCRbuffer,mix.

10. PCR#94:10"@95C;linkto#95.

11. Duringthelast5minsof#94,add45ulofTaq/PCRbuffer,mix(justuncapthetubes,don"ttakethemout;addthroughtheoil)

12. Add45ulmoreoiltocover,flicktubestomakesuretherearenobubblesbefore#95starts.

13. PCR#95:1"@94C,1"@56C,3"@72C;35cycles;linkto#96.

14. PCR#96:5"@72C;linkto#97.

15. PCR#97:soakat4C.

16. RemovetheoilontoParafilm,andPipettesamplesintonewtubes.

17. Load3ulofeachsampleonto1%agarosegel(usingsmallcomb)todefineamountandsizeofproductDNA.

18. StoreremainingDNAPCRproductat-20Cuntilnicktranslation.

Notes

RemembertoincludesufficientnormalDNAreactionsfortheplannedCGH(usuallytwodoublePCRreactions:100ngin100uleach).

Weusuallyincludea50ngMPE-600celllinepositivePCR/CGHcontrol,andaPCRblanknegativecontrol.

ForPCRoffreshDNA(fromunfixedmaterial),50nginputDNAisusedper50ulPCRreaction.Thenuse25ulofthePCRproductforeach50ulnicktranslation.Thenuse20ulofFITCorBiotin-labeledprobe,or15ulofTexasRed-labeledprobe,or10ulofDioxigenin-labeledprobeforeachCGHreaction.

ForPCRofparaffinDNA(fromformalinfixed/paraffinembeddedtissue),itisbesttoaddonly1ulofDNAper50ulPCRreaction.Thereactionmaybeinhibited(lessproduct)iftoomuchoftheDNAextractionsolutionisadded(perhapsbreakdownproductsfromtheProteinaseKreactionareinhibiting).Use40ulofthisPCRproductper50ulnicktranslationreaction,andthenuseallofthenicktranslationproductfortheCGH.Ifthedigoxigenin-labeledprobesizeisgood(>600bp),only35-40uloftheprobestillyieldsgoodCGH.

Wehavefoundthatitisunwisetoamplifymorethan13tubesperPCRreaction.

Materials

PCRMix(10reactions):100ul5XPCRbuffer+400ulddH20.

PreamplificationMix(10reactions):50ulofthePCRmix+1ulTopo-1(0.1ul/sample)+2ulMgCl2(50mM;0.2ul/sample);mixwell.

5XPCRBuffer(Boehringer):500ul10XTaqbuffer;10uldATP,10uldCTP,10uldGTP,10uldTTP,125ulMgCl2(50mM);6.7ulof1.4mMDOPPrimer,335uldH20.Total1000ul(storefrozenin200ulaliquots).

10xTaqBuffer(Boehringer):100mMTrisHCl;2mMMgCl2;500mMKCl;1mg/mlgelatin.

DOPPrimer:5"-CCG-ACT-CGA-GNN-NNN-NAT-GTG-G-3".Useat1.4mM.

Topo-1:Promega.

Sequenase-Vers2(USB/Amersham):Useat1:8withSequenasedilutionbuffer

TaqPolymerase:Boehringer:5U/ul(use2U/sample)

dNTP"s(Boehringer):usestockof100mMeach.

PCRPrograms

#91:30min@37C;Linkto#92.

#92:10"@96C;Linkto#93.

#93:5"@30C,2min@37C,1min@94C,10sec@30C,5cycles;Linkto#94.

#94:10"@95C;Linkto#95.

#95:1"@94C,1"@56C,3"@72C;35cycles;Linkto#96.

#96:5"@72C;Linkto#97.

#97:soakat10C.