METHODS OF RNA AMPLIFICATION IN THE
PRESENCE OF DNA
This application claims the benefit of U.S. Provisional Application Nos. 61/038,696, filed Mar. 21, 2008; 61/074, 991, filed Jun. 23, 2008; and 61/085,811, filed Aug. 1, 2008, which applications are incorporated herein by reference in their entirety. This application is related to the following 10 co-pending international patent applications PCT/US2009/ 033936 and PCT/US2009/033964 filed Feb. 12, 2009, which claim the benefit of the foregoing provisional applications and U.S. Provisional Application No. 61/028,146; filed Feb. 12, 2008; and which applications are all incorporated herein 15 by reference in their entirety.
BACKGROUND OF THE INVENTION
The detection and/or quantitation of specific nucleic acid 20 sequences is an important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various 25 types of treatment. Such procedures are also useful in detecting and quantitating microorganisms in foodstuffs, water, industrial and environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored. Other applications are found 30 in the forensic sciences, anthropology, archaeology, and biology where measurement of the relatedness of nucleic acid sequences has beenused to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, and aid in classifying a variety of life forms. 35
The ability to amplify ribonucleic acid (RNA) is an important aspect of efforts to elucidate biological processes. Amplification of the total cellular mRNAs prepared from any cell or tissue is important for gene expression profiling. Total cellular mRNA represents gene expression activity at a defined 40 time. Gene expression is affected by cell cycle progression, developmental regulation, response to internal and external stimuli and the like. The profile of expressed genes for any cell type in an organism reflects normal or disease states, response to various stimuli, developmental stages, cell differ- 45 entiation, and the like. Non-coding RNAs have been shown to be of great importance in regulation of various cellular functions and in certain disease pathologies. Such RNAs are often present in very low levels. Although analysis of non-amplified mRNA is feasible, a significant amount of starting 50 mRNA can be required. Thus, amplification methods capable of amplifying low abundance RNAs, are of great importance.
RNA amplification is commonly performed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method and variations thereof. These methods are based on 55 replication of RNA by reverse transcriptase to form single stranded DNA complementary to the RNA (cDNA), which is followed by amplification techniques such as polymerase chain reaction (PCR) or linear isothermal amplification to produce multiple copies of single or double stranded DNA, or 60 RNA. However, the total amount of sample RNA that is available is frequently limited by the amount of biological sample from which it is derived. Biological samples are often limited in amount and precious. Moreover, the amount of the various RNA species is not equal; some species are more 65 abundant than others are, and these are more likely and easier, to analyze. The ability to amplify RNA sequences enables the
analysis of less abundant, rare RNA species. The ability to analyze small samples, by means of nucleic acid amplification, is also advantageous for design parameters of large scale screening of effector molecule libraries, for which reduction in sample volume is a major concern both for the ability to perform very large scale screening or ultra high throughput screening, and in view of the limiting amounts of library components. Methods of amplification from RNA templates have been described, for example in U.S. Pat. No. 6,946,251.
RNA in biological samples is often in the presence of DNA. Amplification of the target RNA in the presence of DNA results in unwanted amplification products as described herein. It is desirable to prevent these unwanted products that originate from initiation on DNA because they may interfere with analysis of target RNA amplification products, result in erroneous conclusions and affect the amplification yield from the target RNA. Purification of RNA from DNA results in reduced yield and/or RNA quality. Therefore, it is highly desirable to develop improved amplification methods of target RNA in the presence of DNA. Moreover, the ability to selectively amplify RNA in a sample comprising total nucleic acid from a biological sample will also assist in the development of procedures and methods for selective amplification in situ as well as directly from stabilized cell lysates. This is especially useful when there are minute amounts of sample for analysis.
Therefore, there is a need for improved RNA amplification methods that overcome drawbacks in existing methods. The invention provided herein fulfills this need and provides additional benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
One aspect of the invention comprises a method of generating multiple copies of a polynucleotide sequence of or complementary to target RNA which is in a sample comprising DNA, said method comprising the steps of: (a) hybridizing to the target RNA a first primer comprising a sequence (A) that is not complementary to the target RNA, and a sequence (B) at the 3'-end which hybridizes to the target RNA; (b) extending the first primer with at least one enzyme comprising RNA-dependent DNA polymerase activity in the presence of at least one compound comprising DNA-dependent DNA polymerase inhibitor activity, whereby a complex comprising a primer extension product and the target RNA is produced, whereby the first primer extension product comprises a sequence (Y) that is complementary to the target RNA and comprises sequence (A). In one embodiment of this aspect of the invention, the first primer extension product is marked or labeled with the sequence (A) by the use of the first primer comprising the sequence (A). The method further comprises the steps of: (c) disabling or removing at least one compound comprising DNA-dependent DNA polymerase inhibitor activity; and (d) producing multiple copies of a polynucleotide sequence complementary to the target RNA and/or complementary to sequence (Y) using sequence (A). In one embodiment of this aspect of the invention, the method selectively generates multiple copies of the target RNA polynucleotide sequence of interest in the presence of non-target DNA and/or RNA. In some embodiments, the method is suitable for amplifying one or more whole transcriptomes or a substantial fraction thereof in the presence of DNA. In one embodiment of this aspect of the invention, the method generates amplified polynucleotides suitable for downstream