Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-03
2001-10-09
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S183000, C536S023100, C536S024330
Reexamination Certificate
active
06300069
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the fields of molecular biology. The present invention is directed to novel compositions and methods useful for the generation of nucleic acids from a ribonucleic acid template and further nucleic acid replication. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase-polymerase chain reaction (RT-PCR).
BACKGROUND OF THE INVENTION
The detection, analysis, transcription, and amplification of nucleic acids are the most important procedures in modern molecular biology. The application of such procedures for RNA analysis is especially important in the investigation of gene expression, diagnosis of infectious agents or genetic diseases, the generation of cDNA, and analysis of retroviruses, to name but a few applications. The reverse transcription of RNA, followed by polymerase chain reaction amplification, commonly referred to as RT-PCR or RNA-PCR, has become widely used for the detection and quantification of RNA.
The RT-PCR procedure involves two separate molecular syntheses: (i) the synthesis of cDNA from an RNA template; and (ii) the replication of the newly synthesized cDNA through PCR amplification. RT-PCR may be performed under three general protocols: (1) uncoupled RT-PCR, also referred to as two-step RT-PCR; (2) single enzyme coupled RT-PCR (coupled RT-PCR is also referred to as one-step RT-PCR or continuous RT-PCR), in which a single polymerase is used for both the cDNA generation from RNA as well as subsequent DNA amplification; and (3) two (or more) enzyme coupled RT-PCR, in which at least two separate polymerases are used for initial cDNA synthesis and subsequent replication.
In uncoupled RT-PCR, reverse transcription is performed as an independent step using buffer and reaction conditions optimal for reverse transcriptase activity. Following cDNA synthesis, an aliquot of the RT reaction product is used as template for PCR amplification with a thermostable DNA Polymerase, such as Taq DNA Polymerase, under conditions optimal for PCR amplification.
In coupled RT-PCR, reverse transcription and PCR amplification are combined into a single reaction mixture. Single enzyme RT-PCR utilizes the reverse transcriptase activity of some DNA polymerases, such as Taq DNA Polymerase and Tth DNA polymerase, whereas two-enzyme RT-PCR typically uses a retroviral or bacterial reverse transcriptase (e. g. AMV-RT, MMLV-RT, HIV-RT, EIAV-RT, RAV2-RT, Carboxydothermus hydrogenoformans DNA Polymerase or a mutant, variant or derivative thereof), and a thermostable DNA polymerase (e. g. Taq, Tbr, Tth, Tih, Tfi, Tfl, Pfu, Pwo, Kod, VENT, DEEPVENT, Tma, Tne, Bst, Pho, Sac, Sso, ES4 and others or a mutant, variant or derivative thereof).
Coupled RT-PCR provides numerous advantages over uncoupled RT-PCR. Coupled RT-PCR requires less handling of the reaction mixture reagents and nucleic acid products than uncoupled RT-PCR (e.g., opening of the reaction tube for component or enzyme addition in between the two reaction steps), and is therefore less labor intensive, reducing the required number of person hours. Coupled RT-PCR also requires less sample, and reduces the risk of contamination (Sellner and Turbett, 1998).
Single enzyme coupled RT-PCR, is the simplest RT-PCR procedure to date. This system is expensive to perform, however, due to the amount of DNA polymerase required. In addition, the single enzyme coupled RT-PCR method has been found to be less sensitive than uncoupled RT-PCR (Cusi et al., 1994), and limited to polymerizing nucleic acids of less than one kilobase pair (>1 kpb) in length. Two enzyme RT-PCR systems show increased sensitivity over the single enzyme system generally, even when coupled in a single reaction mixture. This effect has been attributed to the higher efficiency of reverse transcriptase in comparison to the reverse transcriptase activity of DNA polymerases (Sellner and Turbett, 1998).
Although the two enzyme coupled RT-PCR system is more sensitive than the uncoupled protocol, reverse transcriptase has been found to interfere directly with DNA polymerase during the replication of the cDNA, thus reducing the sensitivity and efficiency of this technique (Sellner et al., 1992; Aatsinki et al., 1994; Mallet et al., 1995). A variety of solutions to overcome the inhibitory activity of reverse transcriptase on DNA polymerase have been tried, including: increasing the amount of template RNA, increasing the ratio of DNA polymerase to reverse transcriptase, adding modifier reagents that may reduce the inhibitory effect of reverse transcriptase on DNA polymerase (e.g., non-homologous tRNA, T4 gene 32 protein, sulfur or acetate-containing molecules,), and heat-inactivation of the reverse transcriptase before the addition of DNA polymerase.
All of these modified RT-PCR methods have significant drawbacks, however. Increasing the amount of template RNA is not possible in cases where only limited amounts of sample are available. Individual optimization of the ratio of reverse transcriptase to DNA polymerase is not practicable for ready-to-use reagent kits for one-step RT-PCR. The net effect of currently proposed modifier reagents to relieve reverse transcriptase inhibition of DNA polymerization is controversial and in dispute: positive effects due to these reagents are highly dependent on RNA template amounts, RNA composition, or may require specific reverse transcriptase-DNA polymerase combinations (see, for example, Chandler et al., 1998). Finally, heat inactivation of the reverse transcriptase before the addition of the DNA polymerase negates the advantages of the coupled RT-PCR and carries with it all the disadvantages of uncouple RT-PCR systems discussed earlier.
Because of the importance of RT-PCR applications, a coupled RT-PCR system, in the form of a generalized ready-to-use composition, which exhibits high sensitivity, requires a small amount of initial sample, reduces the amount of practitioner manipulation, minimizes the risks of contamination, minimizes the expense of reagents, is not restricted to the use of specific reaction buffers, and maximizes the amount of nucleic acid end product is needed in the art.
SUMMARY OF THE INVENTION
The present invention is directed to novel compositions and methods useful for the generation of nucleic acids from a ribonucleic acid template and further nucleic acid replication. Specifically, the invention is directed to the generation and amplification of nucleic acids by reverse transcriptase-polymerase chain reaction (RT-PCR), utilizing two or more different polymerases in the presence of a homopolymeric nucleic acid. The presence of the homopolymeric nucleic acid serves to negate inhibition of the DNA polymerase, which occurs when a composition comprising two or more polymerases, at least one of which having reverse transcriptase activity, is used. The compositions and methods of present invention are used to generate and replicate DNA molecules complementary to a portion of an RNA template.
The present invention is directed generally to all reaction mixtures that can be used in the generation and replication of a nucleic acid from an RNA template. One embodiment of the present invention is directed to a composition comprising a homopolymeric nucleic acid and one or more of the following reaction components: a reverse transcriptase, a DNA polymerase, one or more oligonucleotide primers, any one or more nucleotide base, an appropriate buffering agent, a salt solution, or other additives useful in RT-PCR.
The present invention offers several advantages compared to known methods for generating cDNA from an RNA target, including, but not limited to:
permitting coupled RT-PCR, involving one reaction solution and reduced handling of reagents and products;
permitting use of a small initial sample of RNA template;
permitting use of a wide range of different reverse transcriptases;
permitting use of a wide range of different DNA polymerases;
permitting use of a wide range of different reaction mixture buffer and salt solutions, inc
Kang Jie
Korfhage Christian
Löffert Dirk
Missel Andreas
Qiagen GmbH
Sisson Bradley L.
Yankwich Leon R.
Zwicker Kenneth P.
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