Quantitative analysis of gene expression using PCR

Details Quantitative analysis of gene expression using PCR Quantitative analysis of gene expression using PCR

2001-02-01

2003-04-22

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S024310, C536S024320, C536S024330

Reexamination Certificate

active

06551783

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method of quantitative analysis of gene expression that is particularly suited for use in automated assay systems involving fluorescent dyes.
BACKGROUND TO THE INVENTION
Of the systems currently available for automated assay to determine mRNA copy number of target gene transcripts the Perkin Elmer Applied Biosystems® ABI PRISM® 7700 sequence detection system and associated TaqMan® probes are one of the most efficient and effective and most widely used.
The Perkin Elmer System is a PCR (Polymerase Chain Reaction) based system in which a probe labelled with a fluorescent reporter molecule (typically 6-FAM, JOE or VIC—see below) and a quencher dye (e.g. TAMRA—see below) is provided which hybridises to the target nucleic acid between the site of hybridisation of the forward and reverse PCR primers. While intact, any fluorescence emitted from the probe's reporter molecule is absorbed by the quencher.
During the primer extension phase of the PCR, the Taq polymerase displaces the reporter probe from the strand of DNA being amplified, and in doing so the 5′>3′ exonuclease activity inherent in the polymerase cleaves the probe, separating the reporter molecule from the vicinity of the quencher. The increase in fluorescent reporter signal within a reaction is a direct measure of the accumulation of PCR product.
The ABI 7700 has a built in thermal cycler and a laser directed at each sample well in which PCR is performed via bi-directional fibre optic cables. Emitted fluorescence travels through the cables to a detector, where signals from released reporter molecules which fall between 520 and 66 nM are collected every few seconds. The increase in fluorescent reporter signal within a reaction is a direct measure of the accumulation of PCR product. The output of the assay is measured as a normalised reporter value, &Dgr;Rn. The normalised reporter value for any sample is calculated by the ABI 7700 sequence detection software by dividing the reporter fluorescent signal by the signal from a reference reporter (ROX) incorporated in the reaction and deducting a baseline fluorescence signal which is established during the first few cycles of PCR. The &Dgr;Rn values are plotted against thermal cycle number to allow visualisation of the extent of PCR product generation. The starting copy number of a target sequence (Cn) is established by determining the fractional cycle number (Ct) at which a PCR product is first detected—the point at which the fluorescence signal passes above a threshold baseline.
To determine Ct the Sequence Detection application collects data from the first few PCR cycles and calculates the average Rn and the standard deviation of the Rn by a default factor of 10 to define a threshold. Finally, the algorithm searches the data for a point that exceeds the baseline by the value of the threshold. The cycle at which this point occurs is defined as Ct. Ct represents a detection threshold for the Sequence Detector. Ct is dependent on the starting template copy number, and the efficiency of both the DNA amplification the PCR system and the cleavage of the fluorogenic probe. When the starting concentration of a template remains constant as the concentration of other PCR components varies, the most efficient PCR system has the lowest Ct.
Quantification of target is established through comparison of experimental Ct values with a standard curve.
The conventional use of the Perkin Elmer TaqMan® assay system had been limited to the use of single dye probes. However, in recent times the system has been adapted for use of multiple reporter dyes within a single reaction tube to perform a convenient multiplex assay in which the mRNA copy number for more than one target gene transcript can be determined within a single tube. To achieve this there must be more than one primer pair present in the tube which presents a problem if there are differences in the initial copy numbers of the gene sequences to be expressed. The more common gene sequence may well exhaust the reagents within the tube preventing amplification of the less common sequence(s).
Perkin Elmer have sought to overcome this problem by limiting the production of the assay PCR products derived from each of the two or more target or marker gene sequences to ensure that if one of the sequences is more abundant than another there will nonetheless be sufficient reagents remaining in the reaction mix to amplify the less abundant even if its amplification and hence detection does not start until the reaction involving the more abundant transcript has run to completion.
In their instructions on how to implement their multiplexing method, Perkin Elmer advise the user (user Bulletin #2 ABI PRISM 7700 Sequence Detection System of Dec. 11, 1997) that if the user does not know the relative abundance of each RNA to be assayed for, then limiting primer concentrations will need to be defined for each by running a matrix of forward and reverse primer concentrations for each and identifying the concentrations as those that show a reduction in &Dgr;Rn but little effect on Ct (threshold cycle). Such characterisation of limiting primer concentrations is inherently time consuming. However, the only alternative proposed by Perkin Elmer is to use rRNA as one of the target gene transcripts avoiding the need for determining limiting primer concentration based upon the fact that rRNA is known to be extremely abundant and will therefore inevitably be more abundant than the other chosen target mRNA in the assay.
It is one general objective of the present invention to provide a method of multiplexing that avoids the need for characterisation of the limiting primer conditions for each of the gene targets without being restricted to use of rRNA as one of the targets.
SUMMARY OF THE INVENTION
According to the present invention there is provided a method of quantitative analysis of gene expression utilising a respective fluorescent reporter signal for at least a first and a second target gene within a single replication reaction vessel the method comprising:
(i) determining a suitable restricted concentration of primer for replication of the first target gene, wherein the concentration of primer is selected to provide a robust, but limited fluorescent amplification signal; and
(ii) determining a suitable concentration of primer for replication of the second target gene, wherein the concentration of primers is selected to give the most efficient detection of amplification as measured by accumulation of fluorescent signal; and
(iii) utilising the selected primer concentration, determining a suitable concentration of second target gene probe, wherein the concentration of the second target gene probe is selected to provide a fluorescent signal that is at least four times greater than the fluorescent signal of the selected first gene primer concentration.
Suitably the concentration of primer is, in each case, determined for both forwards and reverse primers. As used in step (i) of the method the term “limited” means not maximal and the term robust means strong and readily recognisable.
Preferably in step (ii) the concentration of primer(s) is selected to give the earliest detection of amplification.
Suitably the first gene primer concentrations correspond to &Dgr;Rn of less than 0.3 and preferably the selected target gene probe concentration corresponds to a target fluorescent signal &Dgr;Rn of between 1.5 and 6.
In a more particular aspect, the invention provides a method of quantitation of expression of a first and a second target protein-encoding nucleic acids using a PCR based assay in which amplification of nucleic acid is detected by release of a reporter fluorescent signal which is measured as a normalised reporter value (“&Dgr;Rn”), the method comprising:
providing a limiting concentration of a first primer pair, the first primer pair being adapted for amplification of a desired first target, the limiting concentration being selected to provide a relatively low &Dgr;Rn value as measu

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