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1、双脱氧法测定DNA序列NTP,dNTPsandddNTPsddNTPs2,5,13,169,10,15,191,4,7,8,12,14,203,6,11,17,18ddAddCddGddTSeparateanddisplaytheseDNAfragments.2,5,13,169,10,15,191,4,7,8,12,14,203,6,11,17,18ddAddCddGddTLabelledRadioactiveisotopelabelledPRIMER/TEMPLATEMIXDISTRIBUTEDT
2、OFOURTUBES4dNTPsPLUSDNAPOLYMERASEARADIOACTIVEdNTPONEOFFOURTUBESADDddAASECONDADDddCTHIRDddGFOURTHddTToPerformSequencingTHUS:TUBE1.PRIMER+TEMPLATE+4dNTPs+ddATPTUBE2.PRIMER+TEMPLATE+4dNTPs+ddCTPTUBE3.PRIMER+TEMPLATE+4dNTPs+ddGTPTUBE4.PRIMER+TEMPLATE+4dNTPs
3、+ddTTPALLTUBESCONTAINDNAPOLYMERASESCHEMATICOFATRADITIONALSEQUENCINGGEL AUTORADIOGRAMACGTSEQUENCEISREADFROMBOTTOMTOGIVE5’ACGCTAGGGCTCAA3’Theradioactiveisotope-labelleddi-deoxynucleotidesarereplacedwith4fluorescentderivatives,eachbasespecificitybeinglabel
4、ledadifferentcolour;Thereforeallfourreactionscombined;TheDNAfragmentsarevisualisedbyanautomatedspectroscopeastheypassanobservationTheDNAfragmentsareidentifiedbytheirfluorescentsignals;TheinformationisstoreddirectlyonacomputerAutomatedDNAsequencingCOMBIN
5、EDREACTIONSCOMBINEDREACTIONSFLUORESCENCEDETECTORACTIVATIONLASERThermalcyclesequencing
