Methods for making nucleic acids

Details Methods for making nucleic acids Methods for making nucleic acids

2000-05-08

2003-06-24

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C435S091300, C435S091500, C536S023100, C536S024100, C536S024300

Reexamination Certificate

active

06582936

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of this invention is making nucleic acids.
2. Background
The ability to characterize cells by gene expression provides a wide variety of applications in therapy, diagnostics and bio/medical technology. However, in many of these application, the starting or source material such as stem cells, cancerous cells, identified neurons, embryonic cells, etc. is highly limiting, making it necessary to amplify the targeted mRNA populations. Two existing methods for amplifying mRNA populations suffer from significant limitations. One method, the Brady and Iscove method (Brady et al., 1990, Methods Mol & Cell Biol 2, 17-25), produces only short (200-300 bp), extreme 3′ fragments of mRNAs using a PCR-based method which exponentially amplifies artifacts. A second method, the Eberwine protocol (Eberwine et al. (1992) Proc.Natl.Acad.Sci USA 89, 3010-3014) provides sequential linear amplification steps and is the current method of choice for amplifying mRNA populations from limiting material. Nevertheless, this protocol suffers from a number of deficiencies. For example, the amplified product does not represent full-length aRNA for many endogenous mRNAs, and hence the method is of limited use for generating probes or cDNA libraries.
Relevant Literature
Sippel (1973) Eur.J.Biochem. 37, 31-40 discloses the characterization of an ATP:RNA adenyltransferase from
E. coli
and Wittmann et al. (1997) Biochim.Biophys.Acta 1350, 293-305 disclose the characterization of a mammalian poly(A) polymerase. Gething et al. (1980) Nature 287, 301-306 disclose the use of an ATP:RNA adenyltransferase to polyadenylate the ′3 termini of total influenza virus RNA. Eberwine et al. (1996) U.S. Pat. No. 5,514,545 describes a method for characterizing single cells based on RNA amplification. Eberwine et al. (1992) Proc.Natl.Acad.Sci USA 89, 3010-3014, describe the analysis of gene expression in single live neurons. Gubler U and Hoffman B J. (1983) Gene (2-3), 263-9, describe a method for generating cDNA libraries, see also the more recent reviews, Gubler (1987) Methods in Enzymology, 152, 325-329 and Gubler (1987) Methods in Enzymology, 152, 330-335. Clontech (Palo Alto, Calif.) produces a “Capfinder” cloning kit that uses “GGG” primers against nascent cDNAs capped with by reverse transcriptase, Clontechniques 11, 2-3 (October 1996), see also Maleszka et al. (1997) Gene 202, 39-43.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for making nucleic acids. The general methods comprise the steps of adding a known nucleotide sequence to the 3′ end of a first RNA having a known sequence at the 5′ end to form a second RNA and reverse transcribing the second RNA to form a cDNA. According to one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5′ end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3′ end, and the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence. The resultant cDNA transcript may be single-stranded, isolated from the second RNA and optionally converted to double-stranded cDNA, preferably by a DNA polymerase initiating at a noncovalently joined duplex region. The cDNA may also be transcribed to form one or more third RNAs. In another embodiment, the first RNA is made by amplifying a mRNA by the steps of hybridizing to the poly(A) tail of the mRNA a poly(T) oligonucleotide joined to an RNA polymerase promoter sequence, reverse transcribing the mRNA to form single-stranded cDNA, converting the single-stranded cDNA to a double-stranded cDNA and transcribing the double-stranded cDNA to form the first RNA.
In a particular aspect, nucleic acids are made by converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step contacting the single-stranded DNA with a DNA polymerase having 5′ exonuclease activity under conditions whereby the DNA polymerase converts the-single stranded DNA to the double-stranded DNA, wherein the single-stranded DNA is primed with oligonucleotide primer comprising a sequence complementary to the 3′ end of the single-stranded DNA, and at least one of the 5′ end of the primer and the single-stranded DNA comprises an RNA polymerase promoter joined to an upstream (5′) flanking moiety which protects the promoter from the 5′ exonuclease activity of the DNA polymerase.


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