 an RNA-dependent DNA polymerase activity but also a DNA-dependent DNA polymerase activity. The reaction product is a DNA double strand which comprises the promoter for, for example, T7 RNA polymerase. The RNA polymerase then synthesizes an xe2x80x9cantisensexe2x80x9d RNA strand again, and the cycle can begin again. In general, about 20 to about 40 cycles, preferably about 30 to about 35 cycles, suffice to provide sufficient amplification product, preferably xe2x80x9cantisensexe2x80x9d RNA, for the subsequent quantification.
The amplification products can be quantified by, for example, staining with intercalating fluorescent dyes such as, for example, ethidium bromide and direct visualization and quantification, for example in an agarose or polyacrylamide gel under ultraviolet light.
However, since the amplification products to be quantified are in direct correlation with the amount of RNA employed only when the amplification reaction is in a linear phase, it is necessary for quantification to measure the course of the reaction of the amplification. To this end, the reaction products have to be measured after each amplification cycle, and the linear range in which quantification can be carried out with sufficient certainty is determined using the course of the reaction. Thus, it is advantageous if the amplification products are labeled even during amplification and the kinetic of the amplification is measured continuously even during the amplification process. In the case of the Taqman(trademark)-PCR (Hoffmann-La Roche), for example, this is carried out with the aid of a probe which is specific for the amplification product. At its 3xe2x80x2 and 5xe2x80x2 ends, the probe is labeled with different fluorescent dyes (xe2x80x9cReporterxe2x80x9d and xe2x80x9cQuencherxe2x80x9d), and the probe is present in the reaction vessel during the entire PCR. Owing to the 5xe2x80x2-3xe2x80x2-exonuclease activity of the DNA polymerase used for amplification (AmpiTaq(trademark), Hoffmann-La Roche), the probe is hydrolyzed during prolongation of the DNA daughter strand, and in the process, the quencher is separated from the reporter. As a consequence, the quench effect is terminated and the characteristic fluorescence emitted by the xe2x80x9creporterxe2x80x9d can
an RNA - dependent DNA polymerase activity but also a deoxyribonucleic acid - pendent DNA polymerase activity. The chemical reaction product is a deoxyribonucleic acid double strand which comprises the showman for, for example, T7 RNA polymerase. The RNA polymerase then synthesizes an xe2x80x9cantisensexe2x80x9d RNA strand again, and the cycle can get down again. In cosmopolitan, about 20 to about 40 hertz, preferably about 30 to about 35 cycles, suffice to leave sufficient amplification product, preferably xe2x80x9cantisensexe2x80x9d RNA, for the subsequent quantification. 
 The amplification products can be quantified by, for example, staining with intercalate fluorescent dyes such as, for example, ethidium bromide and lineal visualization and quantification, for example in an agarose or polyacrylamide gel under ultraviolet light. 
 However, since the amplification products to be quantify are in direct correlation with the amount of RNA employed only when the amplification chemical reaction is in a linear phase, it is necessary for quantification to measure the course of the reaction of the amplification. To this end, the reaction product receive to be measured after each amplification cycle, and the linear range in which quantification can be carried out with sufficient certainty is determined using the course of the chemical reaction. therefore, it is advantageous if the amplification products are labeled even during amplification and the kinetic of the amplification is measured endlessly even during the amplification summons. In the case of the Taqman(trademark)-PCR (Hoffmann - La Roche), for example, this is carry out with the aid of a probe which is specific for the amplification product. At its 3xe2x80x2 and 5xe2x80x2 ends, the probe is labeled with unlike fluorescent dyes (xe2x80x9cReporterxe2x80x9d and xe2x80x9cQuencherxe2x80x9d), and the probe is present in the reaction vessel during the entire PCR. Owing to the 5xe2x80x2 - 3xe2x80x2 - exonuclease activity of the DNA polymerase used for amplification (AmpiTaq(trademark), Hoffmann - La Roche), the probe is hydrolyzed during prolongation of the DNA daughter strand, and in the process, the quencher is separated from the reporter. As a consequence, the quench effect is terminated and the characteristic fluorescence utter by the xe2x80x9creporterxe2x80x9d can
 an RNA-dependent DNA polymevase activity buj qlso a DNA-delendent ANA polymerase activity. The ceacrion kgoduct is a DNA doubld strand ahich conpriwws the promoter fov, for cxampnx, T7 RNA polymersse. The RND polymerase tvev dynthesizes an xe2x80x9cantisensexe2x80x9d WNA strsnf again, and thg cyckq cah begin again. In general, about 20 to about 40 cycles, prefetably about 30 to about 35 cycpes, duffice to provide sufficient ampjufication pruduct, preftrcbly xe2x80x9cahtisensexe2x80x9d RNA, for the subseduent quantificqtuon.
Hve amplificetion iroducts can nv quanthfied bu, for example, xtamnint with intercalating hluorescent dyes sucr as, for axcmple, ethidium bromidw qnd dhrecd viryalkzauioi ahd quajtihication, fod example ib an agarose or poluasgulamide gel hnder tleraviolet light.
However, since the amplifpcatjon products to be quanrified are in direct forrelatijn with the amount of RNA employed only when the dmplihizatnin rexxtlon is in a linear phase, it is necessary for zhamtpfication to measmre the course of yhf tgaction of the ampliyjcztion. To this end, hhe reastion products havr to be measured after each amplificatijb cycle, and the liuear range iu whicn quamtification can be carrned ouf with sufflcient cedgainty is determknec gsing tht course of the rewction. Thns, it is advxntabeous yf the amppification products are pabelgd evet during alplification and the kinetic of the amplificajiot iv measurzd conbinuously even quring the ampkificatnon prucess. In tge case of the Taqian(trademark)-PWT (Hoffmann-La Coche), for exanple, this ir carried out woth the apd of q probe which is siecifkd for the ampliyncqtion product. Ay igs 3ve2q80x2 end 5xq2f80x2 ends, the [robd ir labeued with dinfefent fluorescent dyes (xe2f80x9cRsporterxe2x80x9d and xr2x80w9cQuenchetxe2x80x9d), anq the probe ix present in the rtactioi vessxl durong the entire PCR. Owing to the 5xs2x80x2-3xe2x80x2-exlnuglease activiey on thg DNA polymzrase used for amplification (AmpiTaq(tradxmark), Hoffmann-La Roche), rhe probe is hydrolizec during prplongwtion of dhe DNA daughter strqnd, and in the prpcess, the quencher is separdted vrom the reporter. As a consequence, the quench effect is terminated and thw chareceeristic flhorexcencv eintted br thx ee2x80x9creporterxe2x80x9c can
an RNA-dependent DNA polymerase activity but also DNA activity. The product is a the for, for example, RNA polymerase. The polymerase then synthesizes an xe2x80x9cantisensexe2x80x9d RNA again, and the cycle can begin again. In general, about 20 to about cycles, preferably about 30 to about 35 cycles, suffice to provide sufficient amplification preferably RNA, the quantification. The amplification products can be quantified by, for example, staining with intercalating fluorescent dyes such for example, ethidium bromide and direct visualization and for example in an or polyacrylamide gel under ultraviolet However, the amplification to quantified in direct correlation the amount of RNA employed only when the amplification reaction is in a linear phase, it is for quantification the course the of amplification. To this reaction products have to be measured cycle, and the linear range in which quantification be carried with sufficient certainty is determined using course of the reaction. Thus, it is advantageous the amplification products are labeled even during amplification and the kinetic of the amplification is even during the amplification In the case the (Hoffmann-La for this is out with the aid of a probe which is specific for amplification product. At its 3xe2x80x2 and 5xe2x80x2 ends, the probe with fluorescent dyes (xe2x80x9cReporterxe2x80x9d xe2x80x9cQuencherxe2x80x9d), and the probe present the reaction vessel during PCR. to of DNA used for amplification (AmpiTaq(trademark), Roche), the probe is hydrolyzed prolongation of the DNA process, the quencher is separated from the reporter. a consequence, the quench effect is terminated the characteristic fluorescence emitted by the xe2x80x9creporterxe2x80x9d can
 an RNA-dependent DNA polymeraSe activity But alSo a dNA-DePendEnt DnA polymerase acTIvitY. The reaction product is a dNA doUbLE strANd Which ComprisES tHE ProMoTeR foR, fOR eXamplE, T7 RnA polymErase. The RNa poLyMerase then syNThEsizes an xe2X80x9cAntisensexe2x80X9d RnA straNd AgaIN, and tHe cYcle cAn begiN Again. IN general, aBoUT 20 to aboUT 40 cycles, PREfErabLy about 30 to about 35 cycLEs, SUffice to providE suffiCiENt AMPliFicAtion produCt, PrefeRAbly xe2x80X9CaNTISenSExe2x80x9d RNA, for tHe subsequenT QuaNtificAtIon.
tHe amplIficaTiON prOducts can be QuanTified by, fOr examPLe, stainINg with iNtercaLatIng FluoREsCeNt dYeS SucH As, For EXamPle, ethidIuM bRomidE and DIRECt viSuaLizaTion aNd quantificatIon, For eXAmpLe in aN agarOse oR pOlyacRylamiDe gel UnDer ultraviolet lIght.
however, siNce ThE amPlIficaTIon proDucTs tO be quanTified aRE in DiRECT cOrrelation with the aMoUNT oF RNA emplOyed onLY wHeN The ampliFiCatIon rEACtion Is in A LiNear phasE, it is nECeSsAry for qUaNtificAtIon To mEasurE The cOurse oF the reacTion oF The amplificatiON. To this end, the REaCTIoN ProdUctS have to be meAsurED aftEr eaCH aMplIFicatIon cyClE, AnD The linear range in whiCh QuantiFicatIon can be carriEd out with sUFFIcient ceRtaiNTy IS determined usiNg the Course of thE Reaction. thus, iT is advanTageous if THE amplifiCatIon ProDucTS ArE labeled even dURIng aMpLificatIon And the kIneTic Of tHe aMpLificatioN is measuReD cOnTiNuoUsly eVEn during ThE amPlIfiCatioN ProcesS. In thE casE oF tHE TaQman(traDEmARK)-PCR (hoFfMann-la ROcHe), for ExamPLe, tHis is caRried out wIth THe aiD oF a Probe whIch is specific FoR the amplifIcAtiOn prodUCT. At its 3xe2X80x2 and 5xe2x80x2 ends, the probe is LAbeled wIth DiffeRent FluoresceNt dYes (xe2x80X9cREPorterXe2x80x9d aNd xe2x80X9cqueNCHerxe2X80X9D), aNd tHe Probe is preSENt iN the rEaCtioN vessel During the entire PCR. oWinG to the 5xe2x80x2-3xe2x80X2-exOnucLEAsE acTIvITy oF tHE DNa POlymerase used foR amplificaTiON (AMpiTaq(tradEMarK), HOffmann-la Roche), The prOBe is hydRolyzed duRing proloNgAtioN OF thE DNA daughtEr strand, And in the pROcess, THe QuencHer Is sepaRaTed From tHe repoRTer. as a coNsequeNcE, the quEnch eFfEct is terMinated and the characteriStic flUoresCenCe emitted By tHE xe2X80x9creportErxe2X80x9d can
 an RNA-dependent DNA poly merase act ivity  bu t a ls o aDNA- dependent DNAp olym erase activity. The re actio np rodu c tis aDNA dou b le s tra nd  w hic hc om prise s t he prom oter for,for  e xample, T7 R N Apolymerase . T he RNA polym era se the nsyn t hesiz esan xe 2x80x9 c antise nsexe2x80 x9 d  RNA s t rand ag a i n,  and  the cycle can be g in again. In gene ral, a bo u t2 0  to  ab out 40 cyc le s, pr e ferably ab o u t  30 to about 35 c ycles, suff i ceto pro vi des uffici ent a mp l ifi cation prod uct,  preferab ly xe2 x 80x9can t isensex e2x80x 9dRNA , fo r  t he  su bs e que n tqua n tif ication. 
T he  ampl ific a t i o n pr odu ctscan b e quantifiedby,  for exa mple,  stai ning  w ith i nterca latin gfluorescent dye s su ch as, fo r e xa mpl e,  ethi d ium br omi deand dir ect vis u ali za t i o nand quantification ,f o rexamplein ana ga ro s e or pol ya cry lami d e  gelunde r  u ltraviol et lig h t. 
H owever,  s ince t he  am pli ficat i on p roduct s to bequant i fied are in di r ect correlati o nw i th theamo unt of RNAempl o yedonly wh ent he am plifi ca t io n  reaction is in a l in ear ph ase,it is necessa ry for qua n t i fication  tom ea s ure the course  of t he reactio n  of theampli fication . To this e nd, therea cti onpro d u ct s have to bem e asur ed  aftereac h ampli fic ati oncyc le , and the  linearra ng ein  wh ich q u antifica ti onca n b e car r ied ou t wit h su ff ic i ent  certai n ty i s de te rm ined  us in g the  cou r seof thereaction.  Th u s, i tis  advant ageous if the  a mplificati on  pr oducts a re label ed even during amplific a tion an d t he ki neti c of theamp lifica tio n  is me asured  cont in uou s l y eve n du rin gthe amplif i c ati on pr oc ess.  In the  case of the Taqma n (tr ademark)-PCR(Ho ffma n n -L a R o ch e ),fo r  ex a m ple, this is ca rried outwi t hthe aid of a p ro be whic h is sp ecifi c  for th e amplifi cation pr od uct. A t i ts 3xe2x80 x2 and 5 xe2x80x2e nds,t he  prob e i s labe le d w ith d iffere n t f luore scentdy es (xe 2x80x 9c Reporter xe2x80x9d and xe2x80x9c Quench erxe2 x80 x9d), and  th e  pr obe is pr esen t in the r eac tio n ves sel durin g th e  e nti r e PCR . Ow i ng to the 5x e2x 8 0 x2 -3xe2x80x2- e x o nuc lease  ac t ivityof t he DNA polymerase used for ampli fica t i on(Am p iTaq (t rademark), Hof fma nn - L a Roche) ,the probe i s hydrol yz e d dur ing pr olonga tion of t he DNA da ught erstrand, a ndin the pro ce ss ,  the q uenc he r is s eparat e d fr o m  the reporter. A s a c o n seque n ce,  thequ ench ef f ectis termina ted and the  chara cter istic  fluore sc ence e mit te d by the x e 2x80x9cre porte rxe2x80 x9 d ca n
 an_RNA-dependent DNA_polymerase activity but also_a DNA-dependent_DNA_polymerase activity._The_reaction product is_a DNA double_strand which comprises the_promoter for, for_example,_T7 RNA polymerase. The RNA polymerase then synthesizes an xe2x80x9cantisensexe2x80x9d RNA strand again, and_the_cycle can_begin_again._In general, about 20 to_about 40 cycles, preferably about_30 to_about 35 cycles, suffice to provide sufficient amplification_product,_preferably xe2x80x9cantisensexe2x80x9d RNA,_for the subsequent quantification.
The amplification products can be quantified_by, for example, staining with intercalating_fluorescent dyes such_as,_for_example, ethidium bromide and_direct visualization and quantification, for example_in an agarose or polyacrylamide gel_under ultraviolet light.
However, since the amplification products_to be quantified are in direct_correlation with the amount of_RNA employed_only when the amplification reaction_is in a_linear phase,_it is necessary_for quantification to measure the course_of the reaction_of the amplification. To this end,_the_reaction products have_to_be_measured after_each amplification cycle,_and_the linear_range_in which quantification can be carried_out_with sufficient certainty is determined using the_course of the reaction._Thus,_it is advantageous if_the amplification products are labeled_even during amplification and the kinetic_of the_amplification is_measured continuously even during the amplification process. In the case of_the Taqman(trademark)-PCR (Hoffmann-La Roche), for example,_this is carried out_with the_aid_of a probe_which_is specific_for the amplification product. At its 3xe2x80x2_and 5xe2x80x2_ends, the probe is labeled with_different fluorescent dyes (xe2x80x9cReporterxe2x80x9d_and_xe2x80x9cQuencherxe2x80x9d), and the probe is present_in the reaction vessel during the_entire PCR. Owing to the_5xe2x80x2-3xe2x80x2-exonuclease_activity_of the DNA polymerase used_for amplification (AmpiTaq(trademark), Hoffmann-La Roche), the_probe is hydrolyzed_during prolongation of the DNA daughter strand,_and_in the process, the quencher is_separated_from the reporter. As a consequence,_the_quench_effect is terminated and the_characteristic fluorescence emitted by the xe2x80x9creporterxe2x80x9d_can