SE521026C2 - Determining efficiency of a polymerase chain reaction (PCR), useful for clinical diagnosis, comprises estimating the efficiency of PCR in the sample from the dependence of the threshold cycle on the dilution factor for each of the genes - Google Patents

Determining efficiency of a polymerase chain reaction (PCR), useful for clinical diagnosis, comprises estimating the efficiency of PCR in the sample from the dependence of the threshold cycle on the dilution factor for each of the genes

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SE521026C2
SE521026C2 SE0101999A SE0101999A SE521026C2 SE 521026 C2 SE521026 C2 SE 521026C2 SE 0101999 A SE0101999 A SE 0101999A SE 0101999 A SE0101999 A SE 0101999A SE 521026 C2 SE521026 C2 SE 521026C2
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pcr
sample
determining
efficiency
determined
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SE0101999A
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SE0101999D0 (en
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Pierre Aaman
Anders Staalberg
Mikael Kubista
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Canag Diagnostics Ab
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Priority to SE0101999A priority Critical patent/SE521026C2/en
Publication of SE0101999D0 publication Critical patent/SE0101999D0/en
Priority to RU2004100112/13A priority patent/RU2004100112A/en
Priority to PCT/SE2002/001093 priority patent/WO2002099135A1/en
Priority to EP02736406A priority patent/EP1399592A1/en
Priority to BR0210094-0A priority patent/BR0210094A/en
Priority to AU2002309423A priority patent/AU2002309423B2/en
Priority to CA002445099A priority patent/CA2445099A1/en
Priority to MXPA03010959A priority patent/MXPA03010959A/en
Priority to JP2003502244A priority patent/JP2004532641A/en
Publication of SE0101999L publication Critical patent/SE0101999L/en
Publication of SE521026C2 publication Critical patent/SE521026C2/en
Priority to US10/694,979 priority patent/US20040132069A1/en
Priority to US11/617,214 priority patent/US20070184470A1/en

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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Abstract

Determining efficiency of a polymerase chain reaction (PCR), where the number of copies of a nucleic acid sequence in a test sample is determined, comprises amplification of DNA by PCR, registering the number of amplification cycles required to obtain a certain amount of product (CT), and estimating the efficiency of the PCR in the sample from the dependence of CT on the dilution factor. Determining efficiency of a polymerase chain reaction (PCR), where the number of copies of a particular nucleic acid sequence in a test sample is determined, comprises amplification of DNA by PCR of the sample itself, or a diluted stock solution of the sample itself, and one or more controlled dilutions of the sample, and registering the number of amplification cycles required to obtain a certain amount of product (CT), and estimating the efficiency of the PCR in the sample from the dependence of CT on the dilution factor. Independent claims are included for the following: (1) diagnosing and/or classifying a disease by comparing the expression ratio of two genes by determining the ratio of the corresponding mRNAs in a sample; (2) monitoring a diseases progress, where the expressions of two or more genes are compared; (3) making a disease prognosis, where the expressions of two or more genes are compared; (4) comparing the presence of splicing variants of a gene by determining their relative amounts; (5) comparing the activities of alternative promoters by determining the relative amounts of their transcripts; (6) determining the amount of a virus or bacteria in a sample; and (7) diagnostic testing for cancer, including lymphoma, where at lest the kappa:lambda expression is determined

Description

25 30 35 521 026 2 Immunoglobuliners tunga kedjor IgH består av fyra distinkta gensegment: variabel (VH), diversitet (D), länkning (JH) och konstant (CH).1 I andra celler än B lymfocyter är dessa segment separerade med långa regioner mellanliggande DNA. Såväl vanliga som neoplastiska B-celler genomgår en slumpartad rekombination i VH/D/JH delarna av IgH genkomplexet under sin utveckling. I denna process blandas ungefär 50 VH, 30 D och 6 CH gener och förs ihop varvid det mellanliggande DNAt avlägsnas, så att endast en av vardera VH, DH och LH generna bevaras i det färdiga omlagrade segmentet. Dessutom sätts ett flertal slumpvisa baser in mellan DH/LH och VH/DH förbindelserna, så att ytterligare variation erhålls. Den konstanta region i den tunga kedjan är densamma i alla immunoglobuliner. Immunoglobulin heavy chains IgH consists of four distinct gene segments: variable (VH), diversity (D), linkage (JH) and constant (CH) .1 In cells other than B lymphocytes, these segments are separated by long regions intermediate DNA. Both normal and neoplastic B cells undergo a random recombination in the VH / D / JH parts of the IgH gene complex during their development. In this process, approximately 50 VH, 30 D and 6 CH genes are mixed and pooled, removing the intermediate DNA so that only one of each VH, DH and LH genes is retained in the finished rearranged segment. In addition, a plurality of random bases are inserted between the DH / LH and VH / DH compounds, so that further variation is obtained. The constant region of the heavy chain is the same in all immunoglobulins.

Den lätta kedjan i immunoglobulinerna, IgL, består av tre distinkta gensegment: variabel (VL), länkning (JL) och konstant (CL), som kodas av två separata loci: Igk och Igit. i< locus har 40 VK segmentf medan A locus har 31 VL segment? Förhållandet mellan antikroppar innehållande 1< och it kedjorna varierar betydligt bland olika ryggradsdjurfis hos människan är km förhållandet 60:40, medan det hos t.ex. mus är 95:5.The light chain of the immunoglobulins, IgL, consists of three distinct gene segments: variable (VL), linker (JL) and constant (CL), which are encoded by two separate loci: Igk and Igit. i <locus has 40 VK segmentf while A locus has 31 VL segment? The ratio between antibodies containing 1 <and the IT chains varies considerably among different vertebrates in humans, the km ratio is 60:40, while in e.g. mouse is 95: 5.

Friska individer har lymfocyter som alla i princip utsöndrar unika receptorproteiner. Men hos individer som har lymfom eller relaterade lymfoprolifera sjukdomar, börjar en icke- stimulerad klon av lymfocyter dela sig mer eller mindre ohämmat och förekommer till slut i ett så stort antal att den dominerar lymfocytpopulationen med följd att den växelverka även med kroppsegna substanser. Exempel på sådana sjukdomar är lymfoid leukemi, lymfocytisk hyperplasia.....Healthy individuals have lymphocytes that all secrete unique receptor proteins. However, in individuals with lymphoma or related lymphoproliferative diseases, an unstimulated clone of lymphocytes begins to divide more or less uninhibited and eventually occurs in such large numbers that it dominates the lymphocyte population with the result that it interacts with its own substances. Examples of such diseases are lymphoid leukemia, lymphocytic hyperplasia .....

Det traditionella förfarandet att diagnostisera dessa lymfom är genom att studera morfologiska förändringar och immunofenotypiska data.The traditional procedure for diagnosing these lymphomas is by studying morphological changes and immunophenotypic data.

Samoszuk, M. K. Et al, Diagnostic Immunology, 3:133-138 (1985), Limitations of Numerical Ratios for Defining Monoclonality of Immunoglobulin Light Chains in B-cell Lymphomas, visar att med flödescytometri det är möjligt att definiera och studera optimala numeriska kriterier för särskiljning mellan B-cell lymfom och benign hyperplasi på basis av kappazlambda kvot. Man visar att data indikerar att kvoter mindre än 0,7 och större än 5,5 är optimum för särskiljning mellan lymfom och benign hyperplasi, men att det föreligger ett falska negativa svar om 27% och falska positiva svar om 6%. Detta betraktas som relativt liten känslighet. Kappazlambda kvoter anses härvid som relativt specifika, men okänsliga parametrar för att skilja mellan B-cell lymfom och benign lymfoid hyperplasi. 10 15 20 25 30 35 5213 026 Yao-Tseng Chen et al, Modern Pathology, vol. 7, nr 4, 429-434 (1994), Clonality Analysis of B-cell Lymphoma in Fresh-frozen and Paraffin-embedded Tissues: The Effect of Variable Polymerase Chain Reaction Parameters, visar användning av PCR för bestämning av IgH gen omlagringar. Analys sker medelst bestämning av ett 100 till 130 baspar segment som indikerar B-cell närvaro. Denna artikel visar inte på användning av PCR för bestämning av kappa:lambda kvoter för bestämning av lymfom närvaro.Samoszuk, MK Et al, Diagnostic Immunology, 3: 133-138 (1985), Limitations of Numerical Ratios for Defining Monoclonality of Immunoglobulin Light Chains in B-cell Lymphomas, shows that with flow cytometry it is possible to define and study optimal numerical criteria for distinction between B-cell lymphoma and benign hyperplasia on the basis of kappazlambda ratio. It is shown that data indicate that ratios less than 0.7 and greater than 5.5 are the optimum for distinguishing between lymphoma and benign hyperplasia, but that there is a false negative response of 27% and false positive responses of 6%. This is considered relatively low sensitivity. Kappazlambda ratios are considered to be relatively specific, but insensitive parameters for distinguishing between B-cell lymphoma and benign lymphoid hyperplasia. 10 15 20 25 30 35 5213 026 Yao-Tseng Chen et al, Modern Pathology, vol. 7, No. 4, 429-434 (1994), Clonality Analysis of B-cell Lymphoma in Fresh-frozen and Paraffin-embedded Tissues: The Effect of Variable Polymerase Chain Reaction Parameters, demonstrates the use of PCR to determine IgH gene rearrangements. Analysis is done by determining a 100 to 130 base pair segment that indicates B-cell presence. This article does not disclose the use of PCR to determine kappa: lambda ratios to determine lymphoma presence.

Nuovo, G. J., et al, J Histochemistry & Cytochemistry, vol 49(2),:139-145 (2001), In Situ Determination of T-cell Receptor Beta Expression Patterns, visar på användning av omvänt transskriptas (RT) PCR för T-cell neoplasm och polyklonalitet hos reaktiva lymfknutor. Metoden användes också på TCRB gen omlagringar i vävnadssektioner för definitiv diagnos av T-cell vs. B-cell lymfom. Bestämning av kappazlambda kvoter sker inte. Även ett flertal metoder har utvecklats baserade på PCR.Nuovo, GJ, et al, J Histochemistry & Cytochemistry, vol 49 (2) ,: 139-145 (2001), In Situ Determination of T-cell Receptor Beta Expression Patterns, demonstrates the use of reverse transcriptase (RT) PCR for T -cell neoplasm and polyclonality of reactive lymph nodes. The method was also used on TCRB gene rearrangements in tissue sections for definitive diagnosis of T-cell vs. B-cell lymphoma. Kappazlambda quotas are not determined. A number of methods have also been developed based on PCR.

~ En målsättning med föreliggande uppfinning är att överkomma begränsningarna som diskuterades ovan hos de traditionella existerande PCR baserade metoderna.An object of the present invention is to overcome the limitations discussed above of the traditional existing PCR based methods.

Ytterligare målsättningar med föreliggande uppfinning är: ø Att testet ska kunna göras på ett litet material celler o Att testet ska vara robust, särskilt att det ska vara okänsligt för kontamination av andra celltyper. ø att resultat på testet skall kunna erhållas snabbt, företrädesvis i realtid v Att testet skall kunna erhållas med varje vävnadsprov innehållande B-celler eller T- celler.Further objects of the present invention are: ø That the test should be able to be performed on a small material cells o That the test should be robust, in particular that it should be insensitive to contamination of other cell types. ø That results on the test can be obtained quickly, preferably in real time v That the test can be obtained with any tissue sample containing B cells or T cells.

Den konstanta regionen innehåller olika klonalitet och omfattar därvid olika sekvenser, nämligen kappa och lambda sekvenser. Härvid är Igk - kappasekvensen GAACTGTGGC TGCACCATCT GTCTFCATCT TCCCGCCATC TGATGAGCAG TTGAAATCT G GAACT GCCT C TGTTGTGTGC CT GCT GAATA ACTTCTATCC CAGAGAGGCC AAAGTACAGT GGAAGGTGGA TAACGCCCT C CAATCGGGTA ACT CCCAGGA GAGTGTCACA GAGCAGGACA GCAAGGACAG CACCTACAGC CT CAGCAGCA CCCTGACGCT GAGCAAAGCA GACTACGAGA AACACAAAGT CT ACGCCT GC GAAGTCACCC ATCAGGGCCT GAGCTCGCCC GTCACAAAGA GCTTCAACAG GGGAGAGTGT TAGAGGGAGA AGTGCCCCCA CCT GCT CCT C AGTTCCAGCC TGACCCCCT C CCATCCTIT G GCCTCTGACC CTFITTCCAC AGGGGACCT A CCCCTATTGC 10 15 25 30 521 026 4 GGTCCTCCAG CTCATCTITC ACCTCACCCC CCT CCTCCT C CTTGGCI I IA ATTATGCTAA TGTTGGAGGA GAATGAATAA ATAAAGTGAA TCI I IGCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A Primern som användes för att detektera kappasekvensen har företrädesvis i sin framåt riktade sekvens, sekvensen 5'-TGA GCA AAG CAG ACT ACG AGA-3'och i sin återriktade sekvens, sekvensen 5'-GGG GTG AGG TGA AAG ATG AG-3'.The constant region contains different clonality and comprises different sequences, namely kappa and lambda sequences. Hereby Igk - kappa sequence GAACTGTGGC TGCACCATCT GTCTFCATCT TCCCGCCATC TGATGAGCAG TTGAAATCT G GAACT GCCT C TGTTGTGTGC CT GCT GAATA ACTTCTATCC CAGAGAGGCC AAAGTACAGT GGAAGGTGGA TAACGCCCT C CAATCGGGTA ACT CCCAGGA GAGTGTCACA GAGCAGGACA GCAAGGACAG CACCTACAGC CT CAGCAGCA CCCTGACGCT GAGCAAAGCA GACTACGAGA AACACAAAGT CT ACGCCT GC GAAGTCACCC ATCAGGGCCT GAGCTCGCCC GTCACAAAGA GCTTCAACAG GGGAGAGTGT TAGAGGGAGA AGTGCCCCCA CCT GCT CCT C AGTTCCAGCC TGACCCCCT C CCATCCTIT G GCCTCTGACC CTFITTCCAC AGGGGACCT A CCCCTATTGC 10 15 25 30 521 026 4 GGTCCTCCAG CTCATCTITC ACCTCACCCC CCT CCTCCT C CTTGGCI In IA ATTATGCTAA TGTTGGAGGA GAATGAATAA ATAAAGTGAA TCI In IGCAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A primer used for detecting kappa sequence has preferably in its forward sequence, the sequence 5'-TGA GCA AAG CAG ACT ACG AGA-3 'and in its redirected sequence, the sequence 5'-GGG GTG AGG TGA AAG ATG AG-3'.

Den PNA sond som användes för att detektera kappa har företrädesvis sekvensen TO-CC Cl l l l lCC, där TO står för ett färgämne som reagerar vid hybridisering, och som företrädesvis utgöres av ett cyaninfärgämne.The PNA probe used to detect kappa preferably has the sequence TO-CC C11-11CC, where TO represents a dye which reacts with hybridization, and which is preferably a cyanine dye.

IgÅ - iambdasekvensen AGCCCAAGGC TGCCCCCTCG GTCACTCT GT TCCCACCCT C CT CT GAGGAG CTTCAAGCCA ACAAGGCCAC ACT GGTGTGT CT CATAAGTG ACTTCTACCC GGGAGCCGTG ACAGTGGCCT GGAAGGCAGA TAGCAGCCCC GTCAAGGCGG GAGTGGAGAC CACCACACCC TCCAAACAAA GCAACAACAA GTACGCGGCC AGCAGCTACC TGAGCCTGAC GCCTGAGCAG TGGAAGTCCC ACAAAAGCT A CAGCTGCCAG GTCACGCATG AAGGGAGCAC CGTGGAGAAG ACAGTGGCCC CT ACAGAATG 'ITCATAGGTT CT CATCCCT C ACCCCCCACC ACGGGAGACT AGAGCTGCAG GATCCCAGGG GAGGGGTCT C TCCTCCCACC CCAAGGCATC AAGCCCTI' CT CCCT GCACT C AATAAACCCT CAATAAATAT TCTCATTGTC AATCAAAAAA AAAAAAAAAA AA Primern som användes för att detektera iambdasekvensen har företrädesvis i sin framåt riktade sekvens, sekvensen 5'- GAG CCT GAC GCC TGA G - 3' och i sin återriktade sekvens, sekvensen 5'- ATl' GAG GGT 'lTA 'lTG AGT GCA G-3'.IGA - iambdasekvensen AGCCCAAGGC TGCCCCCTCG GTCACTCT GT TCCCACCCT C CT CT GAGGAG CTTCAAGCCA ACAAGGCCAC ACT GGTGTGT CT CATAAGTG ACTTCTACCC GGGAGCCGTG ACAGTGGCCT GGAAGGCAGA TAGCAGCCCC GTCAAGGCGG GAGTGGAGAC CACCACACCC TCCAAACAAA GCAACAACAA GTACGCGGCC AGCAGCTACC TGAGCCTGAC GCCTGAGCAG TGGAAGTCCC ACAAAAGCT A CAGCTGCCAG GTCACGCATG AAGGGAGCAC CGTGGAGAAG ACAGTGGCCC CT ACAGAATG 'ITCATAGGTT CT CATCCCT C ACCCCCCACC ACGGGAGACT AGAGCTGCAG GATCCCAGGG GAGGGGTCT C TCCTCCCACC CCAAGGCATC AAGCCCTI 'CT CCCT GCACT C AATAAACCCT CAATAAATAT TCTCATTGTC AATCAAAAAA AAAAAAAAAAA AA The primer used to detect the sequence of the G , sequence 5'- AT1 'GAG GGT' lTA 'lTG AGT GCA G-3'.

Den PNA sond som användes för att detektera har företrädesvis sekvensen TO-TCTC TCCTCC, där TO står för ett färgämne som reagerar vid hybridisering, och som företrädesvis utgöres av ett cyaninfärgämne.The PNA probe used to detect preferably has the sequence TO-TCTC TCCTCC, where TO represents a dye which reacts with hybridization, and which is preferably a cyanine dye.

För att kunna utnyttja hela potentialen hos PCR baserad diagnostik av Iymfom, krävs metoder som är noggranna, säkra och snabba, samt användarvänliga. Föreliggande uppfinningen uppfyller dessa krav inom rimlig omfattning. 10 5215026 Beskrivning av figurer Fig. 1. Kappa och Lambda förhållanden.In order to be able to utilize the full potential of PCR-based diagnostics of lymphoma, methods that are accurate, safe and fast, as well as user-friendly are required. The present invention meets these requirements to a reasonable extent. 5215026 Description of Figures Fig. 1. Coat and Lambda conditions.

De redovisade grafiska bilderna visar kappa-lambda kurvorna efter en PCR test för detektering av B cell lymfom, varvid Fig. 1A visar förhållandet hos en frisk individ, medan Fig. 1B visar förhållandet hos en individ med B cell lymfom. Som framgår av Fig. 1B är förhållandet kappa-lambda starkt förändrat och förskjutet så att förhållandet är 1:99 hos den sjuka individen, medan det hos den friska individen, Fig. 1A, är 60:40, vilket bekräftar tidigare känd kunskap. 521 026 6 Referenser I Tonegawa, S. (1983) Nature, 302, 575-581. 2 Klein, R., Jaenichen, R. & Zachau, H. G. (1993). Eur. J. Immunol. 23, 3248-3262. 3 Williams, S. C., Blaison, G., Martin, T., Knapp, A. M. & Pasquali, J. L. (1994). J.The plotted graphs show the kappa-lambda curves after a PCR test to detect B cell lymphoma, with Fig. 1A showing the condition of a healthy individual, while Fig. 1B shows the condition of an individual with B cell lymphoma. As can be seen from Fig. 1B, the kappa-lambda ratio is greatly changed and shifted so that the ratio is 1:99 in the sick individual, while in the healthy individual, Fig. 1A, it is 60:40, which confirms previously known knowledge. 521 026 6 References I Tonegawa, S. (1983) Nature, 302, 575-581. 2 Klein, R., Jaenichen, R. & Zachau, H. G. (1993). Eur. J. Immunol. 23, 3248-3262. 3 Williams, S. C., Blaison, G., Martin, T., Knapp, A. M. & Pasquali, J. L. (1994). J.

Moi. Biol. 264, 220-232. 4 Hood, L., Gray, W. R., Sanders, B. G. and Dreyer, W. Y. (1967) Cold Spring Harbour Symp. Quant. Biol. 32, 133-146. 5 Mcmure, K.R. and Rouse, A. M. (1970) Fed. Prcc. 19, 704.Moi. Biol. 264, 220-232. 4 Hood, L., Gray, W. R., Sanders, B. G. and Dreyer, W. Y. (1967) Cold Spring Harbor Symp. Quant. Biol. 32, 133-146. 5 Mcmure, K.R. and Rouse, A. M. (1970) Fed. Prcc. 19, 704.

Claims (13)

10 15 20 25 30 521 026 7 PATENTKRAV10 15 20 25 30 521 026 7 PATENT REQUIREMENTS 1. Förfarande för bestämning av B och/eller T-cell-klonalitet, kännetecknat av, att B- och/eller T-cellernas varianter av konstanta regioner bestämmes med avseende på varianternas inbördes förhållande vad avser numeriskt antal, varvid bestämningen sker efter mångfaldigande medelst PCR-teknik, varvid |< (kappa) : k (lambda) förhållandet bestämmes, varvid kappaprimerns framåt riktade sekvens är: 5'-TGA GCA AAG CAG ACT ACG AGA-3' och dess återriktade sekvens är: 5'-GGG GTG AGG TGA AAG ATG AG-3' och lambdaprimerns framåt riktade sekvens är: 5'- GAG CCT GAC GCC TGA G - 3' och dess återriktade sekvens är: 5'-GGG GTG AGG TGA AAG ATG ÅG-3'.Method for determining B and / or T cell clonality, characterized in that the variants of constant regions of the B and / or T cells are determined with respect to the mutual relationship of the variants with respect to numerical number, the determination taking place after multiplication by PCR technique, wherein the | <(kappa): k (lambda) ratio is determined, wherein the forward sequence of the kappa primer is: 5'-TGA GCA AAG CAG ACT ACG AGA-3 'and its redirected sequence is: 5'-GGG GTG AGG TGA AAG ATG AG-3 'and the forward directed sequence of the lambda primer are: 5'- GAG CCT GAC GCC TGA G - 3' and its redirected sequence is: 5'-GGG GTG AGG TGA AAG ATG ÅG-3 '. 2. Förfarande enligt krav 1, vari immunoglobuliners konstanta regioner bestämmes.The method of claim 1, wherein the constant regions of immunoglobulins are determined. 3. Förfarande enligt krav 1 eller 2, vari den konstanta regionens lätta kedja bestämmes.A method according to claim 1 or 2, wherein the light chain of the constant region is determined. 4. Förfarande enligt krav 1 eller 2, vari den konstanta regionens tunga kedja bestämmes.A method according to claim 1 or 2, wherein the heavy chain of the constant region is determined. 5. Förfarande enligt krav 1, vari PCR-tekniken är realtids-PCR.The method of claim 1, wherein the PCR technique is real-time PCR. 6. Förfarande enligt krav 5, vari realtids-PCR sker med en probe som gör det möjligt att påvisa mängden bildad produkt i realtid.A method according to claim 5, wherein real-time PCR is performed with a probe that makes it possible to detect the amount of product formed in real time. 7. Förfarande enligt krav 6, vari proben utgöres av en PNA probe som hybridiserar mot K konstanta regionen.The method of claim 6, wherein the probe is a PNA probe that hybridizes to the K constant region. 8. Förfarande enligt krav 7, vari PNA probens sekvens är: TO-CC ClTlTTCC, där TO står för ett färgämne.The method of claim 7, wherein the sequence of the PNA probe is: TO-CC ClTlTTCC, wherein TO represents a dye. 9. Förfarande enligt krav 6, vari proben utgöres av en PNA probe som 10 521 026 s hybridiserar mot 1 konstanta regionen.The method of claim 6, wherein the probe is a PNA probe that hybridizes to the constant region for 521,026s. 10. Förfarande enligt krav 9, vari PNA probens sekvens är: TO-TCTC TCCTCC, där To står för ett färgämne.The method of claim 9, wherein the sequence of the PNA probe is: TO-TCTC TCCTCC, wherein To represents a dye. 11. Förfarande enligt krav 10, vari regionerna amplifieras i samma reaktionskärl och produkten påvisas med hjälp av skilda probe-färgämnen.The method of claim 10, wherein the regions are amplified in the same reaction vessel and the product is detected using different probe dyes. 12. Förfarande enligt krav 10, vari färgämnet är ett cyanin-färgämne.The method of claim 10, wherein the dye is a cyanine dye. 13. Metod att bestämma B-cell lymfom, vari ett human-lymfatiskt vävnadsprov utsättes för förfarandet enligt krav 1-12, och varvid B-cell lymfom föreligger då kvoten kzk inte är 60:40.A method of determining B-cell lymphoma, wherein a human lymphatic tissue sample is subjected to the method of claims 1-12, and wherein B-cell lymphoma is present when the ratio kzk is not 60:40.
SE0101999A 2001-06-06 2001-06-06 Determining efficiency of a polymerase chain reaction (PCR), useful for clinical diagnosis, comprises estimating the efficiency of PCR in the sample from the dependence of the threshold cycle on the dilution factor for each of the genes SE521026C2 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
SE0101999A SE521026C2 (en) 2001-06-06 2001-06-06 Determining efficiency of a polymerase chain reaction (PCR), useful for clinical diagnosis, comprises estimating the efficiency of PCR in the sample from the dependence of the threshold cycle on the dilution factor for each of the genes
JP2003502244A JP2004532641A (en) 2001-06-06 2002-06-05 Method of measuring gene expression ratio of key gene
BR0210094-0A BR0210094A (en) 2001-06-06 2002-06-05 Method for measuring the expression ratio of a gene in the standard gene class
PCT/SE2002/001093 WO2002099135A1 (en) 2001-06-06 2002-06-05 Method to measure gene expression ratio of key genes
EP02736406A EP1399592A1 (en) 2001-06-06 2002-06-05 Method to measure gene expression ratio of key genes
RU2004100112/13A RU2004100112A (en) 2001-06-06 2002-06-05 METHOD FOR MEASURING THE RELATIONSHIP OF GENE EXPRESSION OF KEY GENES
AU2002309423A AU2002309423B2 (en) 2001-06-06 2002-06-05 Method to measure gene expression ratio of key genes
CA002445099A CA2445099A1 (en) 2001-06-06 2002-06-05 Method to measure gene expression ratio of key genes
MXPA03010959A MXPA03010959A (en) 2001-06-06 2002-06-05 Method to measure gene expression ratio of key genes.
US10/694,979 US20040132069A1 (en) 2001-06-06 2003-10-28 Method to measure gene expression ratio of key genes
US11/617,214 US20070184470A1 (en) 2001-06-06 2006-12-28 Method to measure gene expression ratio of key genes

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SE0101999A SE521026C2 (en) 2001-06-06 2001-06-06 Determining efficiency of a polymerase chain reaction (PCR), useful for clinical diagnosis, comprises estimating the efficiency of PCR in the sample from the dependence of the threshold cycle on the dilution factor for each of the genes

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