U.S. patent application number 10/746993 was filed with the patent office on 2004-08-05 for method for the efficiency-corrected real-time quantification of nucleic acids.
This patent application is currently assigned to Roche Molecular Systems, Inc.. Invention is credited to Gutekunst, Martin, Sagner, Gregor, Soong, Richie, Tabiti, Karim.
Application Number | 20040153254 10/746993 |
Document ID | / |
Family ID | 26006383 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040153254 |
Kind Code |
A1 |
Sagner, Gregor ; et
al. |
August 5, 2004 |
Method for the efficiency-corrected real-time quantification of
nucleic acids
Abstract
The present invention concerns a method for the quantification
of a target nucleic acid in a sample comprising the following
steps: (i) determination of the amplification efficiency of the
target nucleic acid under defined amplification conditions, (ii)
amplification of the target nucleic acid contained in the sample
under the same defined reaction conditions, (iii) measuring the
amplification in real-time, (iv) quantification of the original
amount of target nucleic acid in the sample by correction of the
original amount derived from step (iii) with the aid of the
determined amplification efficiency. The efficiency correction of
PCR reactions according to the invention for the quantification of
nucleic acids can be used for absolute quantification with the aid
of an external or internal standard as well as for relative
quantification compared to the expression of housekeeping
genes.
Inventors: |
Sagner, Gregor; (Penzberg,
DE) ; Tabiti, Karim; (Poecking, DE) ;
Gutekunst, Martin; (Eberfing, DE) ; Soong,
Richie; (Tutzing, DE) |
Correspondence
Address: |
ROCHE MOLECULAR SYSTEMS INC
PATENT LAW DEPARTMENT
1145 ATLANTIC AVENUE
ALAMEDA
CA
94501
|
Assignee: |
Roche Molecular Systems,
Inc.
|
Family ID: |
26006383 |
Appl. No.: |
10/746993 |
Filed: |
December 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10746993 |
Dec 24, 2003 |
|
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09823711 |
Mar 30, 2001 |
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6691041 |
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Current U.S.
Class: |
702/20 ;
435/6.12 |
Current CPC
Class: |
C12Q 1/6851 20130101;
G16Z 99/00 20190201; C12Q 1/6851 20130101; C12Q 2561/113 20130101;
C12Q 2545/114 20130101; C12Q 2545/113 20130101 |
Class at
Publication: |
702/020 ;
435/006 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2000 |
EP |
00 107 036.6 |
Jul 13, 2000 |
DE |
100 34 209.4 |
Claims
What is claimed is:
1. A method for the quantification of a target nucleic acid in a
sample comprising the steps of: a) determination of the
amplification efficiency of the target nucleic acid under defined
amplification conditions; b) amplification of the target nucleic
acid contained in the sample under the same defined reaction
conditions; c) measuring the amplification in real-time; and d)
quantification of the original amount of target nucleic acid in the
sample by correction of the original amount derived from step c)
with the aid of the determined amplification efficiency.
2. The method of claim 1, wherein the efficiency of the
amplification is determined by: a) preparing a dilution series of
the target nucleic acid; b) amplifying the target nucleic acid
under defined reaction conditions as claimed in claim 1, the
amplification of the nucleic acid being measured in real time; c)
determining a defined threshold value; d) determining the cycle
number at which the signal threshold value is exceeded for each
dilution; e) determining a logarithmic linear function of the copy
number of target nucleic acid used for the amplification as a
function of the cycle number at which the signal threshold value is
exceeded; and f) calculating the amplification efficiency E
according to E=G.sup.-a wherein .sup.a is determined as the first
derivative of the function determined in step e) and G is the base
number of the logarithm.
3. The method of claim 1, wherein the efficiency of the
amplification is determined by: a) preparing a dilution series of
the target nucleic acid; b) amplifying the target nucleic acid
under defined reaction conditions as claimed in claim 1, the
amplification of the nucleic acid being measured in real time; c)
determining a defined signal threshold value; d) determining the
cycle number at which the signal threshold value is exceeded for
each dilution; e) determining a linear function of the cycle number
determined in step d) as a function of a logarithm of the copy
number of target nucleic acid used for the amplification; and f)
calculating the amplification efficiency E according to
E=G.sup.-1/a wherein .sup.a is determined as the first derivative
of the function determined in step e) and G is the base number of
the logarithm.
4. The method of claim 1 wherein the efficiency of the
amplification is determined by: a) preparing a dilution series of
the target nucleic acid; b) amplifying the target nucleic acid
under defined reaction conditions as claimed in claim 1, the
amplification of the nucleic acid being measured in real time; c)
determining a defined signal threshold value; d) determining the
cycle number at which the signal threshold value is exceeded for
each dilution; and e) determining the amplification efficiency as a
function of the amount of target nucleic acid.
5. A method for the quantification of a target nucleic acid in a
sample relative to a reference nucleic acid comprising the steps
of: a) determination of the amplification efficiencies of the
target nucleic acid and of the reference nucleic acid under defined
amplification conditions; b) amplification of the target nucleic
acid contained in the sample and of the reference nucleic acid
contained in the sample under the same defined reaction conditions;
c) measuring the amplification of the target nucleic acid and of
the reference nucleic acid in real time; and d) calculation of the
original ratio of target nucleic acid and reference nucleic acid in
the sample by correction of the ratio derived from step c) with the
aid of the amplification efficiencies determined in step a).
6. The method of claim 5, wherein steps b)-d) are additionally
carried out using a calibrator sample and subsequently the ratio of
the quotients determined for the sample and for the calibrator
sample is determined as a measure for the original amount of target
nucleic acid in the sample.
7. A method for the quantification of a target nucleic acid in a
sample relative to a reference nucleic acid comprising the steps
of: a) determination of the amplification efficiencies of the
target nucleic acid and of the reference nucleic acid under defined
amplification conditions; b) amplification of the target nucleic
acid contained in the sample as well as of the reference nucleic
acid contained in the sample under the same defined amplification
conditions; c) measurement of the amplification of the target
nucleic acid and of the reference nucleic acid in real time; d)
determining a defined signal threshold value; e) determining the
cycle numbers at which the signal threshold value is exceeded in
each case during the amplification of target nucleic acid and
reference nucleic acid; and f) calculating the original ratio of
target nucleic acid and of reference nucleic acid in the sample
according to the following formula
N(T).sub.0/N(R).sub.0=E(R).sub.n(R)/E(T).sub.n(T), in which
N(T).sub.0=the original amount of target DNA present in the sample
N(R).sub.0=the original amount of reference DNA present in the
sample E(R)=the amplification efficiency of the reference nucleic
acid n(R)=the cycle number of the reference nucleic acid measured
in step e) E(T)=the amplification efficiency of the target nucleic
acid n(T)=the cycle number of the target nucleic acid measured in
step e).
8. The method of claim 7, wherein steps b), c), e) and f) are
additionally carried out using a calibrator sample and subsequently
the ratio of the quotients determined for the sample and for the
calibrator sample is determined as a measure for the original
amount of target nucleic acid in the sample.
9. The method of claim 7, wherein the real-time measurement of the
amplification of target nucleic acid and reference nucleic acid in
a sample is carried out in separate reaction vessels.
10. The method of claim 7, wherein the real-time measurement of the
amplification of target nucleic acid and reference nucleic acid in
a sample is carried out in the same reaction vessel using
differently labelled hybridization probes.
11. A method for the quantification of a target nucleic acid in a
sample comprising the steps of: a) determination of the
amplification efficiencies of the target nucleic acid and of an
internal or external standard under defined amplification
conditions; b) amplification of the target nucleic acid contained
in the sample and of the internal or external standard under the
same defined reaction conditions; c) measuring the amplification of
the target nucleic acid and of the standard in real time; and d)
calculating the original copy number in the sample by correction of
the copy number derived from step c) with the aid of the
amplification efficiencies determined in step a).
12. A method for the quantification of a target nucleic acid in a
sample comprising the steps of: a) determination of the
amplification efficiencies of the target nucleic acid and of an
internal or external standard under defined amplification
conditions; b) amplification of the target nucleic acid contained
in the sample as well as of the internal or external standard under
the same defined reaction conditions; c) measurement of the
amplification of the target nucleic acid and of the standard in
real time; d) determining a defined signal threshold value; e)
determining the cycle numbers at which the signal threshold value
is exceeded during the amplification of target nucleic acid and
standard; and f) determining the original copy number N(T).sub.0 of
target nucleic acid in the sample according to the formula
N(T).sub.0=N(S).sub.0*E(S).su- p.n(S)/E(T).sup.n(T), in which
N(S).sub.0=the original amount of standard used E(S)=the
amplification efficiency of the standard n(S)=the cycle number of
the standard measured in step e) E(T)=the amplification efficiency
of the target nucleic acid n(T)=the cycle number of the target
nucleic acid measured in step e).
13. The method of claim 12 using an internal standard, wherein
real-time measurement of the amplification of the target nucleic
acid and internal standard is carried out with differently labelled
hybridization probes.
14. The method of claim 12, wherein the amplified nucleic acids are
detected with the aid of at least one fluorescent-labelled
hybridization probe.
15. The method of claim 14, wherein the amplified nucleic acids are
detected with the aid of FRET hybridization probes, molecular
beacons or TaqMan probes.
16. The method of claim 12, wherein the amplified nucleic acids are
detected with the aid of a DNA-binding dye, preferably with
SybrGreen I.
17. A kit containing agents to carry out the method of claim 12.
Description
[0001] The present application claims priority to co-pending
European Patent Application No. 00107036.6, filed Mar. 31, 2000,
and co-pending German Patent Application No. 10034209.4, filed Jul.
13, 2000, each of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of nucleic acid
quantification with the aid of quantitative real-time PCR.
BACKGROUND OF THE INVENTION
[0003] Methods for the quantification of nucleic acids are
important in many areas of molecular biology and in particular for
molecular diagnostics. At the DNA level such methods are used for
example to determine the copy numbers of gene sequences amplified
in the genome. However, methods for the quantification of nucleic
acids are used especially in connection with the determination of
mRNA quantities since this is usually a measure for the expression
of the respective coding gene.
[0004] If a sufficient amount of sample material is available,
special mRNAs can be quantified by conventional methods such as
Northern Blot analysis or RNAse protection assay methods. However,
these methods are not sensitive enough for sample material that is
only available in small amounts or for genes that are expressed
very weakly.
[0005] The so-called RT-PCR is a much more sensitive method. In
this method a single-stranded cDNA is firstly produced from the
mRNA to be analysed using a reverse transcriptase. Subsequently a
double-stranded DNA amplification product is generated with the aid
of PCR.
[0006] A distinction is made between two different variants of this
method:
[0007] In the so-called relative quantification the ratio of the
expression of a certain target RNA is determined relative to the
amount of RNA of a so-called housekeeping gene which is assumed to
be constitutively expressed in all cells independent of the
respective physiological status. Hence the mRNA is present in
approximately the same amount in all cells.
[0008] The advantage of this is that different initial qualities of
the various sample materials and the process of RNA preparation has
no influence on the particular result. However, an absolute
quantification is not possible with this method.
[0009] Alternatively the absolute amount of RNA used can be
determined with the aid of standard nucleic acids of a known copy
number and amplification of a corresponding dilution series of this
standard nucleic acid. There are two alternatives:
[0010] When using external standards the standard and target
nucleic acid are amplified in separate reaction vessels. In this
case a standard can be used with an identical sequence to the
target nucleic acid. However, systematic errors can occur in this
type of quantification if the RNA preparation to be analysed
contains inhibitory components which impair the efficiency of the
subsequent PCR reaction. Such errors can be excluded by using
internal standards i.e. by amplifying the standard and target
nucleic acid in one reaction vessel. However, a disadvantage of
this method is that standards have to be used that have different
sequences compared to the target nucleic acid to be analysed in
order to be able to distinguish between the amplification of the
standard and target nucleic acid. This can in turn lead to a
systematic error in the quantification since different efficiencies
of the PCR amplification cannot be excluded when the sequences are
different.
[0011] PCR products can be quantified in two fundamentally
different ways:
[0012] a) End point determination of the amount of PCR product
formed in the plateau phase of the amplification reaction
[0013] In this case the amount of PCR product formed does not
correlate with the amount of the initial copy number since the
amplification of nucleic acids at the end of the reaction is no
longer exponential and instead a saturation is reached.
Consequently different initial copy numbers exhibit identical
amounts of PCR product formed. Therefore the competitive PCR or
competitive RT-PCR method is usually used in this procedure. In
these methods the specific target sequence is coamplified together
with a dilution series of an internal standard of a known copy
number. The initial copy number of the target sequence is
extrapolated from the mixture containing an identical PCR product
quantity of standard and target sequence (Zimmermann and
Mannhalter, Bio-Techniques 21:280-279, 1996). A disadvantage of
this method is also that measurement occurs in the saturation
region of the amplification reaction.
[0014] b) Kinetic real-time quantification in the exponential phase
of PCR.
[0015] In this case the formation of PCR products is monitored in
each cycle of the PCR. The amplification is usually measured in
thermocyclers which have additional devices for measuring
fluorescence signals during the amplification reaction. A typical
example of this is the Roche Diagnostics LightCycler (Cat. No. 2
0110468). The amplification products are for example detected by
means of fluorescent labelled hybridization probes which only emit
fluorescence signals when they are bound to the target nucleic acid
or in certain cases also by means of fluorescent dyes that bind to
double-stranded DNA. A defined signal threshold is determined for
all reactions to be analysed and the number of cycles Cp required
to reach this-threshold value is determined for the target nucleic
acid as well as for the reference nucleic acids such as the
standard or housekeeping gene. The absolute or relative copy
numbers of the target molecule can be determined on the basis of
the Cp values obtained for the target nucleic acid and the
reference nucleic acid (Gibson et al., Genome Research 6:995-1001;
Bieche et al., Cancer Research 59:2759-2765, 1999; WO 97/46707; WO
97/46712; WO 97/46714).
[0016] In summary in all the described methods for the
quantification of a nucleic acid by PCR the copy number formed
during the amplification reaction is always related to the copy
number formed of a reference nucleic acid which is either a
standard or an RNA of a housekeeping gene. In this connection it is
assumed that the PCR efficiency of the target and reference nucleic
acid are not different.
[0017] Usually a PCR efficiency of 2.00 is assumed which
corresponds to a doubling of the copy number per PCR cycle (User
Bulletin No. 2 ABI Prism 7700, PE Applied Biosystems, 1997).
[0018] However, it has turned out that the real PCR efficiency can
be different from 2.00 since it is influenced by various factors
such as the binding of primers, length of the PCR product, G/C
content and secondary structures of the nucleic acid to be
amplified and inhibitors that may be present in the reaction
mixture as a result of the sample preparation. This is particularly
relevant when using heterologous reference nucleic acids e.g. in
the relative quantification compared to the expression of
housekeeping genes.
SUMMARY OF THE INVENTION
[0019] The object of the present invention was therefore to provide
methods for the quantification of nucleic acids which overcome the
disadvantages of the prior art as described above. The object of
the present invention was in particular to provide methods for the
quantification of nucleic acids in which a target nucleic acid is
quantified independent of the amplification efficiencies of target
nucleic acid and reference nucleic acid.
[0020] This object is achieved according to the invention by a
method for the quantification of a target nucleic acid in a sample
comprising the following steps:
[0021] a) Determining the amplification efficiency of the target
nucleic acid under defined conditions.
[0022] b) Amplifying the target nucleic acid contained in the
sample under the same reaction conditions.
[0023] c) Measuring the amplification in real-time.
[0024] d) Quantifying the original amount of target nucleic acid in
the sample by correction of the original amount derived from step
c) with the aid of the determined amplification efficiency.
[0025] According to the invention this method can be used for
relative quantification compared to the expression of housekeeping
genes as well as for absolute quantification.
[0026] A first aspect of the invention therefore concerns a method
for quantifying a target nucleic acid in a sample compared to a
reference nucleic acid comprising the following steps:
[0027] a) Determining the amplification efficiencies of the target
nucleic acid and reference nucleic acid under defined amplification
conditions
[0028] b) Amplifying the target nucleic acid contained in the
sample as well as the reference nucleic acid contained in the
sample under the same defined amplification conditions.
[0029] c) Measuring the amplification of the target nucleic acid
and reference nucleic acid in real-time
[0030] d) Calculating the original ratio of target nucleic acid and
reference nucleic acid in the sample by correcting the ratio
derived from step c) with the aid of the amplification efficiencies
determined in step a).
[0031] A second aspect of the present invention concerns a method
for the quantification of a target nucleic acid in a sample
comprising the following steps:
[0032] a) Determining the amplification efficiencies of the target
nucleic acid and of an internal or an external standard under
defined amplification conditions
[0033] b) Amplifying the target nucleic acid contained in the
sample as well as the internal or external standard under the same
defined reaction conditions
[0034] c) Measuring the amplification of the target nucleic acid
and standard in real-time
[0035] d) Calculating the original copy number in the sample by
correcting the copy number derived from step c) with the aid of the
amplification efficiencies determined in step a).
[0036] In all methods the amplification efficiencies are preferably
determined by
[0037] a) preparing a dilution series of the target nucleic
acid
[0038] b) amplifying the target nucleic acid under defined reaction
conditions according to A, the amplification of the nucleic acids
being measured in real-time
[0039] c) setting a defined signal threshold value
[0040] d) determining the cycle number for each dilution at which
the signal threshold value is exceeded,
[0041] e) calculating the amplification efficiency based on the
determined cycle numbers.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The object of the invention is achieved by a method for the
quantification of a target nucleic acid in a sample comprising the
following steps:
[0043] a) Determining the amplification efficiency of the target
nucleic acid under defined conditions
[0044] b) Amplifying the target nucleic acid contained in the
sample under the same reaction conditions.
[0045] c) Measuring the amplification in real-time
[0046] d) Quantifying the original amount of target nucleic acid in
the sample by correcting the original amount derived from step c)
with the aid of the determined amplification efficiency.
[0047] The importance of an efficiency correction will be
illustrated by an error calculation. Table 1 shows a theoretical
calculation of the average percentage error of the determined copy
number in the case of amplification efficiencies that are different
from 2.00 as a function of the respective cycle number. The error
is calculated according to the formula
percentage error=(2.sup.n/E.sup.n-1).times.100
[0048] in which E is the efficiency of the amplification and n is
the respective cycle number at which the percentage error is
determined.
1TABLE 1 PCR efficiency Detection Cycle (n) (E) 10 15 20 25 30 35
2.00 -- -- -- -- -- -- 1.97 16% 25% 35% 46% 57% 70% 1.95 29% 46%
66% 88% 113% 142% 1.90 67% 116% 179% 260% 365% 500% 1.80 187% 385%
722% 1290% 2260% 3900% 1.70 408% 1045% 2480% 5710% 13.000% 29.500%
1.60 920% 2740% 8570% 26.400% 80.700% 246.400%
[0049] The amplification efficiency can be determined by various
methods for example by determining a function with which the
measured signal is determined relative to the amplification of the
target nucleic acid as a function of the cycle time.
[0050] The amplification efficiency is preferably determined by a
method in which
[0051] a) a dilution series of a target nucleic acid is
prepared
[0052] b) the target nucleic acid is amplified under defined
reaction conditions as claimed in claim 1 and the amplification of
the nucleic acid is measured in real-time
[0053] c) a defined signal threshold value is set
[0054] d) for each dilution the cycle number Cp is determined at
which the signal threshold value is exceeded
[0055] e) a logarithmic linear function of the copy number of
target nucleic acid used for the amplification is determined as a
function of the cycle number at which the signal threshold value is
exceeded
[0056] f) the amplification efficiency E is calculated according
to
E=G.sup.-a
[0057] wherein a is determined as the first derivative of the
function determined in step e) and G is the base number of the
logarithm.
[0058] In a similar manner the amplification efficiency can also be
determined by a method in which
[0059] a) a dilution series of the target nucleic acid is
prepared
[0060] b) the target nucleic acid is amplified under defined
reaction conditions as claimed in claim 1 and the amplification of
the nucleic acid is measured in real-time
[0061] c) a defined signal threshold value is set
[0062] d) the cycle number Cp at which the signal threshold value
is exceeded is determined for each dilution
[0063] e) a linear function of the cycle number determined in step
d) is determined as a function of a logarithm of the copy number of
target nucleic acid used for the amplification and
[0064] f) the amplification efficiency E is calculated according
to
E=G.sup.-1/a
[0065] wherein a is determined as the first derivative of the
function determined in step e) and G is the base number of the
logarithm.
[0066] Both preferred procedures have the advantage that a
systematic error cannot occur that results from determining the
amplification efficiency in a phase of the PCR reaction in which
there is no longer an exponential amplification of the target
nucleic acid (plateau phase).
[0067] However, it unexpectedly turned out that under certain
conditions the amplification efficiency can also be dependent on
the original amount of target nucleic acid or it can change during
the first cycles of an amplification reaction that is still in the
exponential phase. A subject matter of the invention is thus also a
method for the efficiency-corrected quantification of nucleic acids
in which the efficiency of the amplification is determined by
[0068] a) preparing a dilution series of the target nucleic
acid
[0069] b) amplifying the target nucleic acid under defined reaction
conditions as claimed in claim 1 and measuring the amplification of
the nucleic acid in real-time
[0070] c) setting a defined signal threshold value
[0071] d) determining the cycle number Cp at which the signal
threshold value is exceeded for each dilution
[0072] e) determining the amplification efficiency as a function of
the amount of target nucleic acid.
[0073] This can for example be achieved by derivation of a
continuously differentiable function F(Cp) of the Cp values as a
function of the original copy number or vice versa.
[0074] The function F(Cp)=log (concentration of the original copy
number) can for example be standardized by mathematical algorithms
such as a polynomial fit of a higher degree. The amplification
efficiency E can then be determined by the equation
E=G.sup.-dF(Cp)/dCp
[0075] in which dF/(Cp) is the derivative of the continuous
function and G is the base number of the logarithm. A polynomial
fit of the 4.sup.th degree has proven to be particularly suitable
within the sense of the invention.
[0076] The efficiency-corrected quantification of nucleic acids
according to the invention can in principle be used for methods for
absolute quantification as well as for methods for relative
quantification.
[0077] Hence a subject matter of the present invention in relation
to relative quantification is also a method for the quantification
of a target nucleic acid in a sample relative to a reference
nucleic acid comprising the following steps:
[0078] a) Determination of the amplification efficiencies of the
target nucleic acid and of the reference nucleic acid under defined
amplification conditions.
[0079] b) Amplification of the target nucleic acid contained in the
sample as well as of the reference nucleic acid contained in the
sample under the same defined amplification conditions.
[0080] c) Measurement of the amplification of the target nucleic
acid and of the reference nucleic acid in real-time.
[0081] d) Calculation of the original ratio of target nucleic acid
and reference nucleic acid in the sample by correcting the ratio
derived from step c) with the aid of the amplification efficiencies
determined in step a).
[0082] Such a method according to the invention eliminates on the
one hand the influence of inhibitors that may be present in the
examined sample and, on the other hand, corrects errors which may
occur as a result of different amplification efficiencies of the
target nucleic acid and reference nucleic acid.
[0083] Steps b) to d) are advantageously carried out in a parallel
mixture containing a so-called calibrator sample. The calibrator
sample is a sample which contains the target nucleic acid and
reference nucleic acid in a defined ratio that is constant for each
measurement. Subsequently the ratio of the quotients determined for
the sample and for the calibrator sample is determined as a measure
for the original amount of target nucleic acid in the sample. This
has the advantage that in addition other systematic errors are
eliminated that are due to differences in the detection sensitivity
of the target nucleic acid and reference nucleic acid. Such
systematic errors can for example occur as a result of different
hybridization properties of the hybridization probes or, in the
case of fluorescent-labelled probes, different excitation
efficiencies, quantum yields or coupling efficiencies of the dye to
the probe. Therefore the sample to be tested and the calibrator
sample must be analysed in each experiment with the same detection
agents i.e. with the same batch of fluorescent-labelled
hybridization probes.
[0084] A special embodiment of relative quantification according to
the invention is a method for the quantification of a target
nucleic acid in a sample relative to a reference nucleic acid
comprising the following steps:
[0085] a) Determination of the amplification efficiencies of the
target nucleic acid and of the reference nucleic acid under defined
amplification conditions
[0086] b) Amplification of the target nucleic acid contained in the
sample and of the reference nucleic acid contained in the sample
under the same defined amplification conditions.
[0087] c) Measurement of the amplification of the target nucleic
acid and of the reference nucleic acid in real time.
[0088] d) Determination of a defined signal threshold value.
[0089] e) Determination of the cycle numbers at which the signal
threshold value is in each case exceeded during the amplification
of the target nucleic acid and the reference nucleic acid.
[0090] f) Calculation of the original ratio of target nucleic acid
and reference nucleic acid in the sample according to the
formula
N(T).sub.0/N(R).sub.0=E(R).sup.n(R)/E(T).sup.n(T), wherein
[0091] N(T).sub.0=the original amount of target DNA present in the
sample
[0092] N(R).sub.0=the original amount of reference DNA present in
the sample
[0093] E(R)=the amplification efficiency of the reference nucleic
acid
[0094] n(R)=the cycle number of the reference nucleic acid measured
in step e)
[0095] E(T)=the amplification efficiency of the target nucleic
acid
[0096] n(T)=the cycle number of the target nucleic acid measured in
step e)
[0097] In this embodiment it is advantageous to carry out steps b),
c), e) and f) with a calibrator sample in order to eliminate
systematic errors due to the detection of amplification products
and subsequently the ratio of the quotients measured for the sample
and for the calibrator sample are determined as a measure for the
original amount of target nucleic acid in the sample.
[0098] The ratio obtained in step f) is calculated according to the
invention as follows:
N(T).sub.n=N(T).sub.0.times.E(T).sup.n(T) (1)
N(R).sub.n=N(R).sub.0.times.E(R).sup.n(R) (2)
[0099] in which N(T).sub.n=the amount of target DNA at the signal
threshold value
[0100] and N(R).sub.n=the amount of reference DNA at the signal
threshold value
[0101] From (1) and (2) it follows that: 1 N ( T ) n N ( R ) n = N
( T ) 0 .times. E ( T ) n ( T ) N ( R ) 0 .times. E ( R ) n ( R ) (
3 )
[0102] From this it follows that: 2 N ( T ) 0 N ( R ) 0 = N ( T ) n
.times. E ( R ) n ( R ) N ( R ) n .times. E ( T ) n ( T ) ( 4 )
[0103] Due to the fact that an identical signal threshold value has
been set for the target and reference nucleic acid this may be
approximated to:
N(T).sub.n=N(R).sub.n.
[0104] Under this condition and starting from equation (4) for the
original ratio of target nucleic acid and reference nucleic acid,
this results in the equation
N(T).sub.0/N(R).sub.0=E(R).sup.n(R)/E(T).sup.n(T) (5)
[0105] However, this assumed approximation does not apply when
target nucleic acid and reference nucleic acid are detected with
different sensitivities. According to the invention it is therefore
particularly advantageous to measure a calibrator sample in a
parallel reaction and to determine the ratio of the quotients
N(T).sub.0/N(R).sub.0 measured for the sample and for the
calibrator sample as a measure for the original amount of target
nucleic acid in the sample.
[0106] This results in the following from equation (4) using the
indices 3 A for the sample to be analysed and K for the calibrator
sample N ( T ) 0 A N ( R ) 0 A / N ( T ) 0 K N ( R ) 0 K = N ( T )
n A .times. E ( R ) n A ( R ) N ( R ) n A .times. E ( T ) n A ( T )
N ( T ) nK .times. E ( R ) nK ( R ) N ( R ) nK .times. E ( T ) nK (
T ) ( 6 )
[0107] Due to the fact that an identical signal threshold value has
been set for the sample to be analysed and for the calibrator
sample and that identical agents are used to detect target and
reference amplicons in the sample and in the calibrator sample, the
ratio of the quotient determined for the sample and for the
calibrator sample is as follows: 4 N ( T ) n A N ( R ) n A / N ( T
) n K N ( R ) n K = 1
[0108] Hence the ratio of the quotients of the sample to be
analysed and the calibrator sample is: 5 N ( T ) 0 A N ( R ) 0 A /
N ( T ) 0 K N ( R ) 0 K = E ( R ) n A ( R ) - n K ( R ) * E ( T ) n
K ( T ) - n A ( T ) ( 7 )
[0109] Consequently a relative value can be obtained in this manner
for the original copy number of target nucleic acid in the sample
in which systematic errors due to different amplification
efficiencies as well as due to different detection sensitivities
have been eliminated. The only requirement for the accuracy of the
determined value is the justified assumption that under absolutely
identical buffer conditions the amplification and detection
efficiencies are also identical in the various reaction
vessels.
[0110] Requirement for all methods according to the invention for
relative quantification is that the amplification efficiency of the
target nucleic acid as well as the amplification efficiency of the
reference nucleic acid are determined. Both of these determinations
are preferably carried out by the methods described above by
determining the cycle number at which a certain signal threshold
value is exceeded.
[0111] In a preferred embodiment of relative quantification the
sample is divided into two aliquots and the real-time measurement
of the amplification of the target nucleic acid and reference
nucleic acid is carried out in separate reaction vessels. This
prevents interference between the amplification reactions of the
target nucleic acid and the reference nucleic acid with regard to
their efficiency for example by competition for deoxynucleotides or
Taq polymerase. Furthermore the target nucleic acid and reference
nucleic acid can be detected with the same detection systems, for
example with the same DNA binding dye.
[0112] Alternatively the real-time measurement of the amplification
of target nucleic acid and reference nucleic acid can be carried
out in one sample in the same reaction vessel using differently
labelled hybridization probes. This is particularly advantageous
when only small amounts of sample material are available because
the number of PCR reactions required is halved in this manner.
[0113] If it is intended to determine the absolute amount of target
nucleic acid to be detected in a sample, then the method for the
quantification of a target nucleic acid in a sample comprises the
steps of:
[0114] a) Determination of the amplification efficiencies of the
target nucleic acid and of an internal or external standard under
defined amplification conditions;
[0115] b) Amplification of the target nucleic acid contained in the
sample as well as of the internal or external standard under the
same defined reaction conditions;
[0116] c) Measurement of the amplification of the target nucleic
acid and standard in real time; and
[0117] d) Calculation of the original copy number in the sample by
correcting the copy number derived from step c) with the aid of the
amplification efficiencies determined in step a).
[0118] The sequences of the target nucleic acid and standard
nucleic acid are advantageously substantially identical. However,
when selecting the sequence for an internal standard it must be
taken into account that the available detection system should be
able to distinguish between the standard and target nucleic acid.
This can for example be achieved by using hybridization probes with
different labels for the detection of the target nucleic acid and
internal standard. Ideally oligonucleotides are used for this as
detection probes which can be used to distinguish between minimal
sequence differences such as point mutations.
[0119] An advantage of using an internal standard is that the
inhibitors present in the sample also influence the amplification
of the standard. Hence differences in the amplification
efficiencies can be minimized.
[0120] In contrast the use of an external standard has the
advantage that the amplification reactions of the target nucleic
acid and standard cannot competitively interfere with one another
with regard to their efficiency. Moreover the amplification
products of the standard and target nucleic acid can be detected in
parallel reactions with the aid of the same detection system for
example with the same hybridization probe. A disadvantage is
possible differences in the PCR efficiencies due to inhibitors in
the sample. However, errors in the quantification caused by this
can be eliminated by the method described in the following:
[0121] In a preferred embodiment for the absolute quantification of
a target nucleic acid in a sample the method according to the
invention comprises the following steps:
[0122] a) Determination of the amplification efficiencies of the
target nucleic acid as well as of an internal or external standard
under defined amplification conditions
[0123] b) Amplification of the target nucleic acid contained in the
sample as well as of the internal or external standard under the
same defined reaction conditions
[0124] c) Measurement of the amplification of target nucleic acid
and standard in real-time
[0125] d) Setting a defined signal threshold value
[0126] e) Determination of the cycle number during the
amplification of target nucleic acid and standard at which the
signal threshold value is exceeded
[0127] f) Determination of the original copy number N(T).sub.0 of
the target nucleic acid in the sample according to the formula
N(T).sub.0=N(S).sub.0*E(S).sup.n(S)/E(T).sup.n(T) (8)
[0128] in which
[0129] N(S).sub.0=the original amount of standard used
[0130] E(S)=the amplification efficiency of the standard
[0131] n(S)=the cycle number of the standard measured in step
e)
[0132] E(T)=the amplification efficiency of the standard
[0133] n(T)=the cycle number of the target nucleic acid measured in
step e).
[0134] In this case like the relative quantification, the
amplification efficiencies of the target nucleic acid and the
internal standard are preferably determined as described by
determining the cycle number at which a certain signal threshold
value is exceeded.
[0135] According to the invention N(T).sub.0 is calculated as
follows:
N(T).sub.n=N(T).sub.0*E(T).sup.n(T)
[0136] and
N(S).sub.n=N(S).sub.0*E(S).sup.n(S)
[0137] Since an identical signal threshold value has been set for
the target and standard nucleic acid this approximates to:
N(T)n=N(S)n
[0138] Hence the original copy number of target nucleic acid
present in the sample is calculated according to the equation
N(T).sub.0=N(S).sub.0*E(S).sup.n(S)/E(T).sup.n(T) (8)
[0139] The invention in particular also concerns those embodiments
of the described methods for the efficiency-corrected
quantification of nucleic acids in which the amplification products
are detected by hybridization probes which can be labelled with a
detectable component in many different ways.
[0140] A prerequisite for the efficiency-corrected determination of
the original amount of a target nucleic acid and for the
determination of the amplification efficiencies per se is to define
signal threshold values and subsequently determine the cycle number
for the respective amplification reaction at which a certain signal
threshold value is reached. The signal threshold value can be
determined according to the prior art in various ways:
[0141] According to the prior art the signal threshold value can
for example be a signal which corresponds to a certain multiple of
the statistical variance of the background signal (ABI Prism 7700
Application Manual, Perkin Elmer).
[0142] Alternatively the cycle number at which the signal threshold
value is exceeded can be determined according to the so-called "fit
point above threshold" method (LightCycler Operator's Manual,
B59-B68, Roche Molecular Biochemicals, 1999).
[0143] In a further embodiment the threshold value can be
determined as a relative value instead of an absolute value when,
independently of the absolute value of the signal, the course of
the amplification reaction is determined as a function of the cycle
number and subsequently the nth derivative is calculated. In this
case exceeding certain extremes can be defined as exceeding a
certain signal threshold value (EP Application No. 0016523.4).
Hence this method of determining the threshold value is independent
of the absolute signal strength of for example a fluorescence
signal. Thus it is particularly suitable for those embodiments in
which the target nucleic acid and reference nucleic acid are
amplified in the same reaction vessel and are detected with the aid
of different fluorescent labels. Methods have proven to be
particularly suitable for the efficiency-corrected quantification
of PCR products in which the maximum of the second derivative is
determined as a measure for the signal threshold value.
[0144] The hybridization probes used for the methods according to
the invention are usually single-stranded nucleic acids such as
single-stranded DNA or RNA or derivatives thereof or alternatively
PNAs which hybridize at the annealing temperature of the
amplification reaction to the target nucleic acid. These
oligonucleotides usually have a length of 20 to 100
nucleotides.
[0145] Depending on the detection format the label can be
introduced on any ribose or phosphate group of the oligonucleotide.
Labels at the 3' and 5' end of the nucleic acid molecule are
preferred.
[0146] The type of label must be detectable in the real-time mode
of the amplification reaction. This is for example in principle
also (but not only) possible with the aid of labels that can be
detected by NMR.
[0147] Methods are particularly preferred in which the amplified
nucleic acids are detected with the aid of at least one
fluorescent-labelled hybridization probe.
[0148] Many test procedures are possible for this. The following
three detection formats have proven to be particularly suitable in
connection with the present invention:
[0149] a) FRET Hybridization Probes
[0150] For this test format 2 single-stranded hybridization probes
are used simultaneously which are complementary to adjacent sites
of the same strand of the amplified target nucleic acid. Both
probes are labelled with different fluorescent components. When
excited with light of a suitable wavelength, a first component
transfers the absorbed energy to the second component according to
the principle of fluorescence resonance energy transfer such that a
fluorescence emission of the second component can be measured when
both hybridization probes bind to adjacent positions of the target
molecule to be detected.
[0151] Alternatively it is possible to use a fluorescent-labelled
primer and only one labelled oligonucleotide probe (Bernard et al.,
Analytical Biochemistry 235, p. 1001-107 (1998)).
[0152] b) TaqMan Hybridization Probes
[0153] A single-stranded hybridization probe is labelled with two
components. When the first component is excited with light of a
suitable wavelength, the absorbed energy is transferred to the
second component, the so-called quencher, according to the
principle of fluorescence resonance energy transfer. During the
annealing step of the PCR reaction, the hybridization probe binds
to the target DNA and is degraded by the 5'-3' exonuclease activity
of the Taq polymerase during the subsequent elongation phase. As a
result the excited fluorescent component and the quencher are
spatially separated from one another and thus a fluorescence
emission of the first component can be measured.
[0154] c) Molecular Beacons
[0155] These hybridization probes are also labelled with a first
component and with a quencher, the labels preferably being located
at both ends of the probe. As a result of the secondary structure
of the probe, both components are in spatial proximity in solution.
After hybridization to the target nucleic acid both components are
separated from one another such that after excitation with light of
a suitable wavelength the fluorescence emission of the first
component can be measured (Lizardi et al., U.S. Pat. No.
5,118,801).
[0156] In the described embodiments in which only the target
nucleic acid or only the reference nucleic acid or an external
standard is amplified in one reaction vessel in each case, the
respective amplification product can also be detected according to
the invention by a DNA binding dye which emits a corresponding
fluorescence signal upon interaction with the double-stranded
nucleic acid after excitation with light of a suitable wavelength.
The dyes SybrGreen and SybrGold (Molecular Probes) have proven to
be particularly suitable for this application. Intercalating dyes
can alternatively be used.
[0157] A subject matter of the invention are also kits that contain
appropriate agents to carry out the method according to the
invention. According to the invention these agents are present in
the kit in various compositions. A kit preferably contains reagents
such as for example a reverse transcriptase for preparing a cDNA,
DNA polymerase for the amplification reaction, specific primers for
the amplification reaction and optionally also specific
hybridization probes to detect the amplification product. As an
alternative polymerases for a single-step RT-PCR reaction can be
present in the kit. It is also possible that a kit according to the
invention contains package inserts or disks containing files with
previously determined amplification efficiencies for defined
amplification conditions. Finally the invention also concerns a kit
which additionally contains further reagents for the synthesis and
labelling of oligonucleotides such as fluorescent NHS-esters or
fluorescent-labelled CPGs. Moreover a kit according to the
invention can optionally also contain a DNA which can be used as an
internal or external standard.
[0158] The invention is further elucidated by the following
examples:
EXAMPLE 1
Amplification of Cytokeratin 20 (CK20) and Porphobilinogen (PBGD)
cDNAs
[0159] RNA was isolated from the cell line HT-29 (ATCC) using a
HighPure-RNA Restriction Kit (Roche Diagnostics GmbH). After
semi-quantitative spectrophotometric determination, the RNA
concentration was adjusted to 100 ng/.mu.l in RNA-free water. Three
serial single dilutions were prepared from this with RNA
concentrations of 10 ng, 1 ng and 100 pg/.mu.l.
[0160] Total cDNA was prepared from these dilutions by reverse
transcription under the following conditions:
[0161] 1.times. AMV reverse transcription buffer
[0162] 1 mM of each deoxynucleoside triphosphate
[0163] 0.0625 mM randomized hexamers
[0164] 10.mu. AMV reverse transcriptase
[0165] 10 .mu.l RNA
[0166] Ad. 20 .mu.l water
[0167] All mixtures were incubated for 10 minutes at 25.degree. C.,
30 minutes at 42.degree. C. and 5 minutes at 95.degree. C. for the
cDNA synthesis. Subsequently they were cooled to 4.degree. C. A
sample containing 10 ng/.mu.l HT29 RNA was used as a
calibrator.
[0168] Afterwards the amplification reaction was carried out which
was measured in real-time in the FRET HybProbe format on a
LightCycler instrument (Roche Diagnostics GmbH). Each reaction
mixture was amplified under the following conditions:
[0169] 1.times. fast start DNA hybridization probes buffer
[0170] 1.times. detection mix
[0171] 2 .mu.l cDNA
[0172] Ad. 20 .mu.l water
[0173] The 1.times. detection mix was composed of 0.5 .mu.M forward
and 0.5 .mu.M reverse primers, each 0.2 .mu.M fluorescein and
LC-Red 640 labelled hybridization probes, 4 mM magnesium chloride
and 0.005% Brij-35.
[0174] Primers having SEQ ID NO:1 and SEQ ID NO:2 were used to
amplify a CK20 sequence. The CK20 product was detected using a
fluorescein probe having SEQ ID NO:3 and a LC-Red 640 hybridization
probe having SEQ ID NO:4. Primers having SEQ ID NO:5 and 6 were
used to detect the PBGD sequence. PBGD was detected using a
fluorescein-labelled hybridization probe having SEQ ID NO:7 and an
LC-Red 640-labelled hybridization probe having SEQ ID NO:8.
[0175] The reaction mixtures were firstly incubated for 10 minutes
at 95.degree. C. in the presence of 5 mM magnesium chloride for the
amplification. The actual amplification reaction was carried out
for 50 cycles according to the following scheme:
[0176] 10 sec. 95.degree. C.
[0177] 10 sec. 60.degree. C.
[0178] 5 sec. 72.degree. C.
[0179] After each incubation at 60.degree. C. a fluorescence
measurement was carried out according to the manufacturer's
instructions. The signal threshold value (Cp value) was determined
as the maximum of the 2.sup.nd derivative of the amplification
reaction as a function of the cycle number.
EXAMPLE 2
Determination of the Efficiency of the Amplification of CK20 and
PBGD
[0180] A function was established to determine the efficiency in
which the cycle number Cp determined for the respective
concentration was determined as a function of the decadic logarithm
of the RNA concentration used.
[0181] A linear function was calculated from this function by
regression analysis with the aid of the LightCycler software.
Starting from this function the efficiency was determined according
to the equation
efficiency=10.sup.-1/a
[0182] wherein a is the gradient (1.sup.st derivative) of the
determined regression line.
2 TABLE 2 Conc (ng) Log (ng) Cp-CK20 Cp-PBGD 0.1 -1.0 35.73 38.73 1
0.0 30.13 33.59 10 1.0 24.20 28.63 Efficiency: 1.491 1.578 Cp:
measured cycle number
[0183] The results obtained for CK20 and PBGD are shown in Table 2.
The result shows that on the one hand the efficiencies are
considerably different from 2.00 i.e. a doubling of the target
nucleic acid does not take place with each PCR cycle. On the other
hand, the result shows that the efficiencies of the amplification
of CK20 and PBGD differ significantly from one another under
otherwise identical conditions.
EXAMPLE 3
Determination of the Original Ratio of Target Nucleic Acid and
Reference Nucleic Acid with and Without Correction of the
Amplification Efficiency
[0184] Under the conditions described in Example 1 the ratio
determined of the original amount of CK20 and PBGD should be
independent of the respective amplified concentration of the sample
material used. Hence the determination of the ratio for various
amounts of sample RNA was used to check the effect of an efficiency
correction on the basis of the measured values that were obtained.
In this case the ratio of CK20 to PBGD was determined according to
the invention according to equation (5). On the one hand, the ratio
was determined using the efficiencies obtained from example 2 and
on the other hand with an assumed amplification efficiency of 2.00
for CK20 and for PGD. The results are shown in Table 3:
3TABLE 3 N(T).sub.0/N(R).sub.0 HT29 CP Cp N(T).sub.0/N(R).sub.0
Efficiency (ng) CK20 PBGD Efficiency = 2 corrected 0.1 ng 35.73
38.73 8.00 29.68 1 ng 30.13 33.59 11.00 26.66 10 ng 24.20 28.63
21.56 29.66 M: 13.52 28.66 SD: 7.12 1.74 % CV: 52.7% 6.1% Cp =
measured cycle number M = mean SD = standard deviation % CV =
coefficient of variation
[0185] As can be seen from the table, the efficiency-corrected
values calculated for the ratio of N(T).sub.0/N(R).sub.0 have a
significantly lower standard deviation for the various amounts of
sample RNA than the uncorrected values and a coefficient of
variation of 6.1% compared to 52.7%.
EXAMPLE 4
Efficiency-Correction when Using a Calibrator
[0186] Analogously to Examples 1 and 2 amplification reactions were
carried out in the presence of 10 mM magnesium chloride. In this
case an efficiency of 1.491 was determined for CK20 and an
efficiency of 1.578 was determined for PBGD. In addition the Cp
values of a calibrator sample containing an unknown amount of HT-29
RNA was determined at 5 mM and 10 mM magnesium chloride. The
measured data were used to determine the quotients of the ratios of
CK20 to PBGD between the samples analysed in each case and the
appropriate calibrator according to equation (7). This
determination was carried out on the one hand with an assumed
efficiency of 2 for the amplification of CK20 and PBGD as well as,
on the other hand, with the aid of experimentally determined
amplification efficiencies. The result is shown in Table 4.
4TABLE 4 T:R/C HT29 Cp Cp T:R/C Efficiency MgCl.sub.2 (ng) CK20
PBGD Efficiency corrected 5 mM 0.1 ng 36.59 39.09 0.76 0.92 5 mM 1
ng 30.60 32.60 0.54 0.72 5 mM 10 ng 25.19 27.95 0.91 0.95
calibrator Cal. 24.78 27.67 1.00 1.00 10 mM 0.1 ng 35.73 38.73 0.39
1.04 10 mM 1 ng 30.13 33.59 0.53 0.93 10 mM 10 ng 24.20 28.63 1.04
1.04 calibrator Cal. 24.01 28.38 1.00 1.00 M: 0.70 0.93 SD: 0.26
0.10 % CV: 36.7% 11.1%
[0187] 6 T : R / C = N ( T ) 0 A N ( R ) 0 A / N ( T ) 0 K N ( R )
0 K
[0188] Cp=measured cycle number
[0189] M=mean
[0190] SD=standard deviation
[0191] % CV=coefficient of variation
[0192] As can be seen in Table 4, the efficiency-corrected values
have a lower standard deviation (0.10) as well as a three-fold
lower coefficient of variation than the T:R/C values with an
assumed PCR efficiency of 2.00. This result shows that an
efficiency correction according to the invention is also
advantageous in quantifications in which a standardization with the
aid of calibrators has already been carried out.
EXAMPLE 5
Absolute Quantification of Plasmid DNA
[0193] A decadic dilution series of a plasmid containing the PSA
gene of 10.sup.9 to 10.sup.2 copies was prepared for this purpose.
At the same time a second decadic dilution series with a plasmid
containing the gene for TNF (tumour necrosis factor) with an
unknown copy number of plasmid DNA was prepared. Afterwards the PSA
reaction mixtures were amplified on a LightCycler (Roche
Diagnostics) under standard conditions using the primers having SEQ
ID NO:9 and 10 and the TNF reaction mixtures were amplified using
the primers having SEQ ID NO:11 and 12 (Roche Diagnostics
LightCycler SybrGreen Mastermix, 5 mM final concentration
MgCl.sub.2, 0.5 .mu.M final concentration of each primer). The
amplification was measured in real-time using the DNA binding agent
SybrGreenI (Molecular Probes) under standard conditions in which
the evaluation was carried out according to the manufacturer's
instructions in the second derivative mode.
[0194] The original copy number of the TNF plasmid was determined
in two different ways on the basis of the obtained data.
[0195] On the one hand a calibration line based on the PSA
amplification was generated assuming the same amplification
efficiency for PSA and TNF.
[0196] On the other hand the original copy number was determined
according to formula (8). Analogously to example 2 the
amplification efficiency for PSA and TNF was determined by
calculating a regression line according to the formula
E=10.sup.-1/a
[0197] wherein .sup.a denotes the increase (1.sup.st derivative) of
the calculated regression line. In this case an amplification
efficiency of 2.03 was determined for PSA and an amplification
efficiency of 2.13 was determined for TNF.
[0198] The results of the two different quantification procedures
are shown in Table 5. A so-called dilution check was carried out as
a measure for the accuracy of the determination. The values denoted
dilution check are calculated from the quotients of the copy
numbers measured for the respective dilution of two dilution
mixtures that differ from one another by a factor of 10. Thus a
value of 10.00 would be expected as the ideal value.
5 TABLE 5 Not efficiency corrected Efficiency corrected Determined
Determined copy number copy number Dilution per dilution Dilution
check per dilution Dilution check 1 30826128 10.10 27728632 12.12
10.sup.-1 3053000 13.82 2287050 14.98 10.sup.-2 220900 7.19 152643
7.94 10.sup.-3 30710 11.61 19227 13.89 10.sup.-4 2646 8.55 1384
9.52 10.sup.-5 309.5 7.61 145.4 8.76 10.sup.-6 40.66 3.86 16.6 3.84
10.sup.-7 10.54 4.3 Mean: 8.96 10.16
[0199] As the result of the dilution check from Table 5 shows, the
mean of the efficiency-corrected data results in a value of 10.16,
whereas the mean of non-efficiency-corrected data results in a
value of 8.96 which is considerably further away from the ideal
value of 10.00. From this it follows that an efficiency correction
is also advantageous for embodiments in which an absolute
quantification of nucleic acids with the aid of PCR is carried
out.
[0200] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims. All publications cited
herein are incorporated by reference in their entirety.
* * * * *