Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-17
2001-07-10
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091210, C435S091510, C536S024330
Reexamination Certificate
active
06258543
ABSTRACT:
FIELD OF INVENTION
This invention relates to the general fields of molecular biology, biochemistry, genetics and biological research, and specifically, relates to methods for the quantitative determination of relative amount and precise copy number of messenger RNA transcripts in biological samples. This method, thus, further relates to commercial applications in the areas of medical and biological research and medical diagnostics.
BACKGROUND AND PRIOR ART
Nucleic acid probe technology has been discovered for its value in the detection of various diseases, organisms or genetic defects, and thus, has developed quite rapidly. Current techniques provide the sensitivity lacking in earlier assays and necessary to qualitatively determine the presence of minute quantities of nucleic acid. That is, they are sensitive enough to be able to detect a single copy of a specific nucleic acid sequence. The use of nucleic acid probe tests based on hybridization for quantitative analysis is hindered by a lack of sensitivity.
A number of methodologies involving amplification of nucleic acids are currently used for the measurement of gene expression. The most sensitive of these methodologies utilizes the polymerase chain reaction (PCR) technique, the details of which are provided in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, and U.S. Pat. No. 4,965,188, all to Mullis et al., all of which are specifically incorporated herein by reference. The details of PCR technology, thus, are not included herein. Recently, additional technologies for the amplification of nucleic acids have been described, most of which are based upon isothermal amplification strategies as opposed to the temperature cycling required for PCR. These strategies include, for example, Strand Displacement Amplification (SDA)(U.S. Pat. Nos. 5,455,166 and 5,457,027 both to Walker; Walker et al. (1992) PNAS 89:392; each of which is specifically incorporated herein by reference) and Nucleic Acid Sequence-Based Amplification (NASBA)(U.S. Pat. No. 5,130,238 to Malek et al.; European Patent 525882 to Kievits et al.; both of which are specifically incorporated herein by reference). Each of these amplification technologies are similar in that they employ the use of short, deoxyribonucleic acid primers to define the region of amplification, regardless of the enzymes or specific conditions used.
Generally, amplification of DNA molecules utilizes a thermostable polymerase, for example, Taq polymerase, described in European Patent 258017, specifically which is incorporated herein by reference. Until recently, RNA amplification required a separate, additional step involving the use of non-thermostable reverse transcriptase enzymes to generate a cDNA capable of being amplified by a thermostable DNA polymerase, such as Taq. The discovery of a recombinant thermostable enzyme (rTth) capable of coupling reverse transcription of the RNA with DNA amplification in a single enzyme:single reaction procedure greatly simplified and enhanced RNA amplification (see, Myers & Gelfand (1991)
Biochemistry
30:7661-7666; U.S. Pat. No. 5,407,800 to Gelfand and Myers, both of which are specifically incorporated herein by reference). Recombinant thermostable enzyme polymerase chain reaction (rTthPCR) is exquisitely sensitive.
All PCR-based methods for measuring gene expression have certain features in common: the nucleic acids from a chosen sample must be extracted, and, the target sequence is amplified by PCR, allowing detection and quantitation of that sequence. However, some notable difficulties are encountered when applying PCR to the quantitative measurement of gene expression. The efficiencies of both the extraction step and the amplification step directly affect the outcome of the assay. To determine the number of original copies, the efficiency of the nucleic acid extraction, as well as the efficiency of each PCR reaction must be known. Further, the detection step reveals how many copies of the target sequence have been made, but not how many copies were contained in the original sample An absolute quantitation of sample copy number is difficult using the current state of extraction technologies—if not impossible. The best that can be achieved on a routine basis is quantifying changes in gene expression rate, rather than quantifying the exact numbers of copies of the target sequence contained in the sample. However, this quantification of gene expression rate changes can only be done if the efficiencies of the extraction and amplification processes are constant or can be controlled. There are two basic types of control: the first is commonly known as exogenous control (Gilliland et al. (1990)
PCR Protocols
, Innis et al. ed., pp. 60-69, Academic Press; Wang et al. (1989)
Proc. Natl. Acad. Sci. USA
86:9717-9721, both of which are specifically incorporated herein by reference), and the second, is known as endogenous control (Dveksler et al. (1992)
PCR Methods and Applications
6:283-285; Spanakis (1993)
Nucleic Acids Research
21:3809-3819, both of which are specifically incorporated herein by reference).
Exogenous control involves the use of an artificially introduced nucleic acid molecule that is added, either to the extraction step or to the PCR step, in a known concentration. The concept of adding an exogenous nucleic acid at a known concentration in order to act as an internal standard for quantitation was introduced by Chelly et al. (1988)
Nature
333:858-860, which is specifically incorporated herein by reference. This approach was improved following the realization that PCR amplification efficiency is dependent upon the primer sequences (WO 91/02817 to Wang et al., which is specifically incorporated herein by reference). Therefore, utilizing a control fragment that is amplified with the same primers as the target sequence more accurately reflects target sequence amplification efficiency relative to the internal standard (see, for example, WO 93/02215 to Fox and Griffiths; WO 92/11273 to McCallum et al.; U.S. Pat. No. 5,213,961 to Bunn et al.; and, U.S. Pat. No. 5,219,727 to Wang et al., all of which are specifically incorporated herein by reference). Similar strategies have proven effective for quantitative measurement of nucleic acids utilizing isothermal amplification reactions such as NASBA (Kievits et al., supra) or SDA (Walker, supra).
The use of an endogenous control regulates variations in extraction efficiency. Control choice is important in that several requirements must be met in order for it to work. The first requirement is that the copy number of the control must remain constant; the second. Requirement is that the control must amplify with similar efficiency to the sequence being monitored. Several constitutively expressed genes have been considered as control candidates, since the expression of these genes is relatively constant over a variety of conditions. The most common of these are the &bgr;-actin gene, the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), and the 16S ribosomal RNA gene. While these genes are considered to be constitutively expressed, they are not ideal for the above-discussed purpose. The first problem is that under some conditions the expression level of these genes varies (Spanakis (1993) supra; Dveksler et al. (1992) supra) possibly invalidating them as controls. Further, these genes necessitate using a different set of PCR primers from any other gene being assayed, possibly leading to differences in PCR efficiency, and thus, invalidating them as controls. Zenilman et al. ((1995)
Analytical Biochemistry
224:339-346, which is specifically incorporated herein by reference) attempted to design a competitive polymerase chain reaction method that altered the cDNA generated during reverse transcription using a composite primer to introduce a common intron primer sequence into the cDNA, such that both genomic DNA and cDNA could then be amplified with the same primer set. That method requires careful design of the composite primers so that, following reverse transcription, the melting temperature (Tm)
Gerdes John C.
Marmaro Jeffrey M.
Hogan & Hartson LLP
Myers Carla J.
O'Rourke Sarah S.
Petersen Steven C.
Xtrana, Inc.
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