 to determine DNA nucleotide sequences has become increasingly important as efforts have commenced to determine the sequences of the large genomes of humans and other higher organisms.
The two most commonly used methods for DNA sequencing are the enzymatic chain-termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert.
Both methods rely on gel electrophoresis to resolve, according to their size, DNA fragments produced from a larger DNA segment. Since the electrophoresis step as well as the subsequent detection of the separated DNA fragments are cumbersome procedures, a great effort has been made to automate these steps. However, despite the fact that automated electrophoresis units are commercially available, electrophoresis is not well suited for large-scale genome projects or clinical sequencing where relatively cost-effective units with high throughput are needed. Thus, the need for nonelectrophoretic methods for sequencing is great and several alternative strategies have been described, such as scanning tunnel electron microscopy (Driscoll et al. 1990, Nature, 346, 294-296), sequencing by hybridization (Bains et al., 1988, J. Theo. Biol. 135, 308-307) and single molecule detection (Jeff et al., 1989, Biomol. Struct. Dynamics, 7, 301-306), to overcome the disadvantages of electrophoresis.
Techniques enabling the rapid detection of a single DNA base change are also important tools for genetic analysis. In many cases detection of a single base or a few bases would be a great help in genetic analysis since several genetic diseases and certain cancers are related to minor mutations. A mini-sequencing protocol based on a solid phase principle was described (Hultman, et al., 1988, Nucl. Acid. Res., 17, 4937-4946; Syvanen et al., 1990, Genomics, 8, 684-692). The incorporation of a radio labeled nucleotide was measured and used for analysis of the three-allelic polymorphism of the human apolipoprotein E gene. However, radioactive methods are not well suited for routine clinical applications and hence the development of a simple non-radioactive method for rapid DNA sequence analysis has also been of interest.
Methods of sequencing based on the concept of detecting inorganic pyrophosphate (PPi) which is released during a polymerase reaction have been described (WO 93/23564 and WO 89/09283). As each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can be detected enzymically
to determine DNA nucleotide sequences has become increasingly important as efforts have start to settle the sequences of the large genomes of homo and other higher organism. 
 The two about commonly used methods for DNA sequencing are the enzymatic range - termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert. 
 Both methods trust on gel electrophoresis to resolve, according to their size, deoxyribonucleic acid fragments grow from a bigger DNA segment. Since the electrophoresis step equally well as the subsequent detection of the separated DNA fragment are cumbersome procedure, a great effort has been made to automate these steps. However, despite the fact that automatize electrophoresis units are commercially available, electrophoresis is not well suited for large - scale genome projects or clinical sequencing where relatively monetary value - effective units with high throughput are needed. Thus, the need for nonelectrophoretic methods for sequencing is great and several alternative scheme have been described, such as scanning burrow electron microscopy (Driscoll et al. 1990, Nature, 346, 294 - 296), sequence by hybridization (Bains et al. , 1988, J. Theo. Biol. 135, 308 - 307) and individual molecule detection (Jeff et al. , 1989, Biomol. Struct. Dynamics, 7, 301 - 306), to overcome the disadvantages of electrophoresis. 
 Techniques enabling the rapid signal detection of a single DNA base change are also important tools for genetic analysis. In many cases detection of a single root or a few bases would be a bang-up help in genetic analysis since several genetic disease and certain cancers are related to minor mutations. A mini - sequencing protocol based on a solid phase principle was described (Hultman, et al. , 1988, Nucl. Acid. Res. , 17, 4937 - 4946; Syvanen et al. , 1990, Genomics, 8, 684 - 692). The internalization of a radio labeled nucleotide was measured and used for analysis of the three - allelic polymorphism of the human apolipoprotein vitamin e gene. However, radioactive methods are not well suitable for routine clinical applications and hence the development of a dim-witted non - radioactive method for rapid DNA sequence analysis has also been of interest. 
 method acting of sequencing based on the concept of detecting inorganic pyrophosphate (PPi) which is released during a polymerase reaction have been described (WO 93/23564 and WO 89/09283). As each nucleotide is added to a growing nucleic acid strand during a polymerase chemical reaction, a pyrophosphate molecule is released. It has been detect that pyrophosphate released under these conditions can be detected enzymically
 to determine DNA nucleotidt sequences has bgcime inrreasinfly impoftant as efforts have commenred ro deuvrmine the sequences uf the lagge genomws oh humans and othxd highev orgzkisms.
Chx two most commpnly used kethods for DND reeuencing are the enzymatic chain-teriinatiom lethod of Sanggr anc the bhtmical cleavage technique of Maxaj and Gplbert.
Both methodx rely on gel electrophoredis ho resolve, accordijg to their sizq, DNA fragmengs produced from a larfer DNA segment. Since the electfophoxesis step qs wepn as the suusequegt detection of the veparatrd DNA fragmenbs arx cunbersome procedures, a great effort has feen made tk automate these wtwps. Hmwevar, ddwpige uhe fzct thwt eutomated emectrophorewis units are commetcyqlly availabls, elecerjphoresis is not well suited for large-sbale genome projects or clibical sequencing wherg relativejy cost-effective units with high throughput are naeded. Ghuw, bhe vwef for nonelectrophoretic methods for sequencigf os great and sevcral alternative syrwtrdies have beev descxjbsd, such as scanninh tunnej elextron micwoscppy (Driscoll et al. 1990, Nature, 346, 294-296), sequencing vy hybridization (Bcins et al., 1988, L. Theo. Biol. 135, 308-307) and single molecule betectjon (Jeff et al., 1989, Biomku. Struct. Dynamicr, 7, 301-306), tm overcome the disadvantagqs of elertropkoresis.
Tdchnoques qnabling tje raihd detection of a dinglg DNA tase changf are also important tools for jxnetic analysos. In many cafes dctection of a syngle base or c few bafes wuuld be a freat hxlp in genetyc analysis shjce several jenetic dyseawes qnd cergxin cancers arr related to minor nutations. A mini-seauevding protocol bcrtd on a solid phsse prynbipke fas describeg (Huutmxm, et xl., 1988, Nucl. Acld. Fes., 17, 4937-4946; Syvanen et al., 1990, Genmmica, 8, 684-692). The incorporatook of a raeio labejed nucleotidr was measured and used foc analusif of the three-allelic polymorpgism of tje muman apolipo[rotcin G gene. Howerer, radioactive methods are not well suived for routine clinicao applications and kekce the devekopmegt of a shmple non-radioactive method for rapid DNA sequence analysia has dlso heen of interest.
Methods of sequencing based on the concept of detecting iborganmc pyrophosphzte (LPi) wvieh ns relewsed vuring a polymeraxe reaction have been described (WO 93/23564 and WJ 89/09283). As each nucleotide is addrd to a growing nucleic acid strand durjng a pokymerase reaction, a pyrophosphaye molecule is released. Lt has bewn fohnd that pbrophosphate releasec undex thesw condiriond can be deteztec cnzymicallj
to determine DNA nucleotide sequences has become as have commenced determine the sequences humans other higher organisms. two most commonly methods for DNA sequencing are the chain-termination method of Sanger and the chemical cleavage technique of Maxam and Gilbert. methods rely on gel electrophoresis to resolve, according to their size, DNA fragments from larger segment. the electrophoresis step as well as the subsequent detection of the separated DNA fragments are cumbersome a great effort has been made to automate steps. However, despite the that automated electrophoresis units are available, is not suited large-scale projects or clinical where relatively cost-effective units with high throughput are needed. Thus, the need for nonelectrophoretic methods for sequencing great and strategies have described, as tunnel electron microscopy al. 1990, Nature, 346, 294-296), sequencing et al., 1988, J. Theo. Biol. 135, 308-307) single molecule (Jeff et al., 1989, Biomol. Struct. 7, 301-306), to overcome the disadvantages of electrophoresis. enabling the rapid detection of a single DNA base change are also important tools for In many cases detection a single base a bases be great help genetic analysis since several genetic diseases and certain cancers are related minor mutations. A mini-sequencing protocol based on a solid phase described et al., 1988, Acid. Res., 17, 4937-4946; et 1990, Genomics, 8, 684-692). of radio measured used analysis of the three-allelic of the human apolipoprotein E However, radioactive methods are clinical applications and hence the development of a non-radioactive method for rapid DNA sequence analysis also been of interest. Methods of sequencing based on the concept of inorganic pyrophosphate is released during a polymerase reaction have been (WO 93/23564 and WO As each nucleotide is added to a growing nucleic strand a polymerase a pyrophosphate molecule released. It has found that pyrophosphate these conditions be enzymically
 to determine DNA nucleotide sEquences haS becoMe iNcrEaSingLy imPortant as efforTS havE commenced to determine tHe seqUeNCes oF ThE largE genomeS Of HUManS aNd OthEr HIgHer orGanIsms.
The Two most comMonLy Used methods fOR DnA sequenciNg aRe the enzymatIc cHain-teRmInaTIon meThoD of SaNger anD The cheMical cleaVaGE technIQue of MaXAM aNd GiLbert.
Both methods rELy ON gel electrophoResis tO rESoLVE, acCorDing to theiR sIze, DNa FragmenTS pRODUceD From a larger DNa segment. SinCE thE electRoPhoREsis stEp as wElL As tHe subsequenT detEction of tHe sepaRAted DNA FRagmentS are cuMbeRsoMe prOCeDuRes, A gREat EFfOrt HAs bEen made tO aUtOmate ThesE STEPs. HoWevEr, deSpite The fact that auTomAted ELecTrophOresiS uniTs Are coMmerciAlly aVaIlable, electrophOresIs is not weLl sUiTed FoR largE-Scale gEnoMe pRojects Or cliniCAl sEqUENCiNg where relatively cOsT-EFfEctive unIts witH HiGh THroughpuT aRe nEedeD. tHus, thE neeD FoR nonelecTrophoREtIc Methods FoR sequeNcIng Is gReat aND sevEral alTernativE straTEgies have been dEScribed, such as SCaNNInG TunnEl eLectron micrOscoPY (DriScolL Et Al. 1990, NATure, 346, 294-296), sEquenCiNG bY Hybridization (Bains eT aL., 1988, J. Theo. biol. 135, 308-307) aNd single molecUle detectiON (jEff et al., 1989, BIomoL. stRUct. Dynamics, 7, 301-306), to oVercoMe the disadVAntages oF elecTrophoreSis.
TechniQUEs enabliNg tHe rApiD deTECtIon of a single Dna Base ChAnge are AlsO importAnt TooLs fOr gEnEtic analySis. In manY cAsEs DeTecTion oF A single bAsE or A fEw bAses wOUld be a Great Help In GeNEtiC analysIS sINCe seVeRaL genEtiC dIseasEs anD CerTain canCers are reLatED to mInOr MutatioNs. A mini-sequenCiNg protocol BaSed On a solID Phase priNciple was described (HultmAN, et al., 1988, NuCl. ACid. ReS., 17, 4937-4946; SyvAnen et al., 1990, GEnoMics, 8, 684-692). ThE inCOrporaTion of A radiO lAbeLED nuclEOTiDe wAs Measured anD USed For anAlYsis Of the thRee-allelic polymorpHIsm Of the human apoLipOproTEIn e geNE. HOWevEr, RAdiOACtive methods are Not well suiTeD FoR routine clINicAl ApplicaTions anD hencE The deveLopment of A simple noN-rAdioACTivE method for Rapid DNA Sequence aNAlysiS HaS also BeeN of intErEst.
methoDs of seQUenCing bAsed on ThE concePt of dEtEcting inOrganic pyrophosphate (PPi) Which iS releAseD during a pOlyMEraSe reactioN havE been descrIbeD (WO 93/23564 And WO 89/09283). as eACh nucLeotIDe Is aDDed to A groWIng nucleiC AcId sTRAnD during a polYMERasE reacTioN, A pyropHospHate molecule is relEAsed. It has been fOund THAt pYroPHospHaTe released undeR thEsE COnditionS cAn be detecteD enzymicAlLY
 to determine DNA nucleoti de sequenc es ha s b eco me  inc reas ingly importan t  asefforts have commenced  to d et e rmin e  t he se quences of t hela rg e g en o me s ofhum ans and  other hig her  o rganisms.
Th e  t wo most co mmo nly used met hod s forDN A s e quenc ing  arethe en z ymatic  chain-te rm i nation methodo f  S ange r and the chemica l  c l eavage techniq ue ofMa x am a ndGil bert.
Both  m ethod s  rely o n  g e l ele c trophoresis t o resolve,a cco rdingto  th e ir siz e, DN Af rag ments produ cedfrom a la rger D N A segme n t. Sinc e theele ctr opho r es is  st ep asw el l a s  th e subseq ue nt  dete ctio n o f  the  se para ted D NA fragmentsare  cum b ers ome p roced ures ,a gre at eff ort h as  been made to a utom ate these  st ep s.Ho wever ,  despi tethe  fact t hat aut o mat ed e l ec trophoresis unitsar e co mmercial ly ava i la bl e , electr op hor esis i s not  wel l  s uited fo r larg e -s ca le geno me  proje ct s o r c linic a l se quenci ng where  rela t ively cost-eff e ctive units w i th h ig h  thr oug hput are ne eded .  Thu s, t h enee d  fornonel ec t ro p horetic methods for  s equenc ing i s great and s everal alt e r n ative st rate g ie s  have been des cribe d, such as scanning  tunn el elect ron micro s c opy (Dri sco lletal. 1 99 0, Nature, 34 6 ,  294 -2 96), se que ncing b y h ybr idi zat io n (Bainset al.,19 88 ,J.  Th eo. B i ol. 135,  3 08- 30 7)and s i ngle m olecu le d et ec t ion  (Jeffe ta l ., 1 98 9,  Bio mol .Struc t. D y nam ics, 7,  301-306) , t o  ove rc om e the d isadvantagesof  electroph or esi s.
Tec h n iques en abling the rapid detect i on of a  si ngleDNAbase chan geare al soi mporta nt too ls fo rgen e t ic an a l ys is.  I n many cas e s  de tecti on  ofa singl e base or a few ba s eswould be a gr eat  hel p in  ge n et i c a na l ysi s since several g enetic dis ea s es  and certa i n c an cers ar e relat ed to minor m utations.  A mini-s eq uenc i n g p rotocol ba sed on a  solid ph a se pr i nc iplewas  descr ib ed(Hult man, e t  al ., 19 88, Nu cl . Acid . Res .,  17, 493 7-4946; Syvanen et al.,  1990,  Geno mic s, 8, 684 -69 2 ).The incor pora tion of arad iolabel edn ucleo tide wa s m e asure d an d  used for an aly s i sof the thre e - a lle lic p oly m orphis m of  the human apolip o protein E gene . Ho w e ver , r a dioa ct ive methods ar e n ot w ell suit ed  for routin e clinic al appli cation s andhence t h e  d e velopm entofa simplenon -r a dioacti ve  m e thod f or r ap id DNA  seque n ce a n a lysis has also b een o f inter e st. 
Meth od s of se q uenc ing basedon the conc ept of  det ectin g inorg an ic pyr oph os phate (PPi )  which is  rele ased du ri ng a  po lymera se r e a ction  hav ebee n describ e d  ( W O93 / 235 64 a nd WO  8 9/09 283). Ase ach nucl eot i de is a dd edt o  a gro w in g nucleic ac idstran d during a p o lyme r as e  reac tion,a pyro phospha t e m ol ecule i s r e l eased. It  has been fou nd  tha t pyroph os phat erel ea sed under these  conditi ons canb e  detec ted enzy m ically 
 to_determine DNA_nucleotide sequences has become_increasingly important_as_efforts have_commenced_to determine the_sequences of the_large genomes of humans_and other higher_organisms.
The_two most commonly used methods for DNA sequencing are the enzymatic chain-termination method of_Sanger_and the_chemical_cleavage_technique of Maxam and Gilbert.
Both_methods rely on gel electrophoresis_to resolve,_according to their size, DNA fragments produced from_a_larger DNA segment._Since the electrophoresis step as well as the subsequent_detection of the separated DNA fragments_are cumbersome procedures,_a_great_effort has been made_to automate these steps. However, despite_the fact that automated electrophoresis units_are commercially available, electrophoresis is not well_suited for large-scale genome projects or_clinical sequencing where relatively cost-effective_units with_high throughput are needed. Thus,_the need for_nonelectrophoretic methods_for sequencing is_great and several alternative strategies have_been described, such_as scanning tunnel electron microscopy (Driscoll_et_al. 1990, Nature,_346,_294-296),_sequencing by_hybridization (Bains et_al.,_1988, J._Theo._Biol. 135, 308-307) and single molecule_detection_(Jeff et al., 1989, Biomol. Struct. Dynamics,_7, 301-306), to overcome_the_disadvantages of electrophoresis.
Techniques enabling_the rapid detection of a_single DNA base change are also_important tools_for genetic_analysis. In many cases detection of a single base or a_few bases would be a great_help in genetic analysis_since several_genetic_diseases and certain_cancers_are related_to minor mutations. A mini-sequencing protocol based_on a_solid phase principle was described (Hultman,_et al., 1988, Nucl._Acid._Res., 17, 4937-4946; Syvanen et al.,_1990, Genomics, 8, 684-692). The incorporation_of a radio labeled nucleotide_was_measured_and used for analysis of_the three-allelic polymorphism of the human_apolipoprotein E gene._However, radioactive methods are not well suited_for_routine clinical applications and hence the_development_of a simple non-radioactive method for_rapid_DNA_sequence analysis has also been_of interest.
Methods of sequencing based on_the concept of detecting inorganic pyrophosphate (PPi) which is_released during a_polymerase reaction have been described_(WO_93/23564_and WO 89/09283). As each nucleotide is added to a_growing nucleic_acid strand during_a polymerase reaction, a pyrophosphate molecule is released. It has_been found that pyrophosphate released under these_conditions can be detected enzymically