Simple and efficient method to label and modify...

Details Simple and efficient method to label and modify... Simple and efficient method to label and modify...

1998-10-16

2001-05-29

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C536S023100, C536S024300

Reexamination Certificate

active

06238865

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to 3′-terminus modified nucleic acids including DNA and RNA, and methods for modifying 3′-termini of nucleic acid compounds.
2. Description of the Related Art
Labeling RNA 3′-termini is important for studies of gene regulation at RNA level and RNA biological function in vitro or in vivo, and for clinical test and research. There are two commonly used methods for end-labeling RNA: 3′-labeling with 3′,5′-[5′-
32
P]-pCp and T4 RNA ligase (England, et. al., Nature, 275:560-561, 1978; Uhlenbeck, et. al., The Enzymes, Vol. XV:31-58, 1982), and 3′-labeling with [&agr;-
32
P]-cordycepin 5′-triphosphate (CoTP or 3′-deoxy-ATP) and poly(A) polymerase (Linger, et. al., Nucleic Acids Res., 21:2917-2920, 1993; Edmonds, et. al., The Enzymes, Vol. XV:217-239, 1982). The 3′-labeling methods are less satisfactory. Labeling with T4 RNA ligase requires high concentrations of pCp and enzyme, and is less efficient at labeling long RNAs (Linger, et. al., Nucleic Acids Res., 21:2917-2920, 1993). Poly(A) polymerase labels short RNAs poorly (Linger, et. al., Nucleic Acids Res., 21:2917-2920,1993) and also requires a high concentration of [&agr;-
32
P]-CoTP for optimal incorporation (Beltz, et. al., Fed. Proc., 41:1450-1455,1982).
SUMMARY OF THE INVENTION
We have developed a new method for 3′-terminus labeling or modifying nucleic acid including RNA based on the principle of synthesis of Okazaki Fragments (Okazaki, et. al., Proc. Natl. Acad. Sci. USA, 64:1242-1248, 1969). Our method allows to introduce a single nucleotide to RNA 3′-termini. It is a simple and efficient method to label or modify RNA 3′-termini using DNA polymerase and a synthetic template with defined overhang nucleotides. The ready availability of short synthetic oligodeoxynucleotides should allow any RNA of known sequence to be extended in a template-directed manner at its 3′-terminus, and therefore selectively labeled, by DNA polymerase in the presence of the appropriate dNTP (FIG.
1
). After screening a number of polymerases, we found that DNA polymerase I large fragment [Klenow fragment (Gubler, et. al., Methods Enzymol., 152:330-335, 1987; Sanger, et. al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977)] is capable of rapidly and efficiently incorporating [&agr;-
32
P]-dATP onto RNA 3′-termini.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.


REFERENCES:
patent: 5266466 (1993-11-01), Tabor et al.
England et al., Nature 275: 560-561 (1978).*
Linger et al., 3′-end Labeling of RNA with Recombinant Yeast Ploy(A) Polymerase, Nucleic Acids Research 21(12): 2917-2920 (1993).*
The Gibco-BRL Catalog pp. 375-377 and 383-387 (1992 Edition).*
Chirala et al., Gene 47: 297-300 (1996).*
Huang et al., Nucleic Acids Research 24(21): 4360-4361 (1996).*
LeBlond et al., Analytical Biochemistry 177: 413-418 (1989).*
Oyama et al., Analytical Biochemistry 172: 444-450 (1988).

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