Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-09
2004-04-20
Fredman, Jeffrey (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100
Reexamination Certificate
active
06723509
ABSTRACT:
TECHNICAL FIELD
The field of this invention is nucleic acids, particularly nucleic acid labeling techniques, and more particularly ribonucleic acid labeling techniques.
BACKGROUND OF THE INVENTION
The end-labeling of ribonucleic acids is fundamental to a variety of molecular biology applications. Applications in which RNA is end-labeled include: hybridization assays, in which the probe employed is an end-labeled ribonucleic acid of the subject invention, e.g. Southern analyses, northern analyses, DNA library screens, in situ hybridization experiments, e.g. chromosome squashes, tissue sections, etc.; sequencing applications, e.g. RNA direct chemical sequencing methods; hybridizations of labeled RNAs to chips/arrays; and the like.
A number of different protocols have been developed for 3′-end labeling RNA. In a first method that has been reported in the literature, bacteriophage T4 RNA ligase is used to attach radioactively labeled ribonucleotides to the 3′ end of RNA molecules. Such ligase reactions typically require long incubation times to achieve sufficient labeling, e.g. 12 hours. In another method, terminal deoxynucleotidyl transferase (TdT) is employed to attach labeled deoxyribonucleotides to the 3′ ends of RNAs. Disadvantages of this method include the fact that TdT cannot label with ribonucleotides (i.e. it cannot use ribonucleotides as donors) and that TdT uses both DNA and RNA as a substrate. Finally, poly(A) polymerase has been employed to label RNAs with radioactively labeled ribonucleotides. Disadvantages with this protocol include the use of radioactively labeled materials.
As such, despite the number of different protocols that have been developed for the 3′ end-labeling of ribonucleic acids, there continues to be interest in the development of new methods for end-labeling ribonucleic acids. Of particular interest would be the development of a method of end-labeling ribonucleic acids which was able to rapidly and efficiently label a ribonucleic acid with non-radioactively labeled ribonucleotides, particularly fluorescently labeled ribonucleotides.
RELEVANT LITERATURE
U.S. Patents of interest include: U.S. Pat. Nos. 5,525,497 and 5,573,913. Also of interest are: Linger et al., Nuc. Acids Res. (1993) 21:2917-2920; Martin & Keller, RNA (1998) 4:226-230; Wiessman, S. M., METHODS OF DNA & RNA SEQUENCING, (Praeger, 1983) pp261-304; Richardson & Gumport, Nuc. Acids Res. (1983) 11:6167-6184; Rosemeyer et al., Anal. Biochem. (1995) 224: 446-449; Sippel, J. Biochem. (1973) 37:31-40; and Winter & Brownlee, Nuc. Acids Res. (1978) 5:3129-3139.
SUMMARY OF THE INVENTION
Methods and kits are provided for end-labeling ribonucleic acids with non-radioactive labels, particularly fluorescent labels. In the subject ribonucleic acid end-labeling methods, ribonucleic acid is contacted with non-radioactively labeled ribonucleotides, e.g. fluorescently labeled ribonucleotide, under conditions sufficient for covalent attachment of the labeled ribonucleotides to the 3′ end of the ribonucleic acid to occur. The subject methods and kits find use in a variety of applications in which the end-labeling of ribonucleic acid with a non-radioactive label is desired.
DEFINITIONS
The term “nucleic acid” as used herein means a polymer made up of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.
The terms “ribonucleic acid” and “RNA” as used herein means a polymer that includes at least one ribonucleotide at its 3′ end, e.g. a polymer made up completely of ribonucleotides.
The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer made up of deoxyribonucleotides.
The term “oligonucleotide” as used herein denotes single stranded nucleotide multimers of from about 10 to 100 nucleotides and up to 200 nucleotides in length.
The term “polynucleotide” as used herein refers to single or double stranded polymer composed of nucleotide monomers of generally greater than 100 nucleotides in length.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods are provided for end-labeling ribonucleic acids with non-radioactive labels. In the subject methods, ribonucleic acid is contacted with non-radioactively labeled ribonucleotides, e.g. fluorescently labeled ribonucleotide, in the presence of a prokaryotic poly(A) polymerase under conditions sufficient for covalent attachment of one or more of the labeled ribonucleotides to the 3′ end of the ribonucleic acid to occur. Also provided are kits for practicing the subject methods. In further describing the subject invention, the subject methods will be discussed first in greater detail followed by a description of the kits for practicing the subject methods.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
It must be noted that as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
Methods
As summarized above, the subject invention is directed to methods of end-labeling ribonucleic acids with non-radioactive labels. In the broadest sense, ribonucleic acids that may be end-labeled according to the methods of the subject invention are nucleic acids that have at least one ribose sugar at their 3′ end with a free 3′ hydroxyl group. The ribonucleic acids can be any desired chemically or/and enzymatically synthesized nucleic acid, e.g. a nucleic acid produced in vivo by a cell, which, apart from the 3′-terminal ribonucleotide, can contain any nucleotide units i.e. in particular deoxyribonucleotide or/and ribonucleotide units. However, nucleic acid acceptor molecules are preferred which have at least two and in particular at least three ribose sugars at their 3′ end. Nucleic acid molecules are particularly preferred which are composed of more than 50% and essentially exclusively of ribonucleotide units i.e. ribonucleic acids, where ribonucleic acids of particular interest include either fragmented or unfragmented total RNA, polyA+RNA, and the like.
The ribonucleic acids are sufficiently long to be end-labeled according to the subject methods. Typically, the ribonucleic acids are at least 10 nt long, usually at least 20 nt long and more usually at least 30 nt long, where the ribonucleic acids may be significantly longer, e.g. the size of full length mRNA transcripts, etc.
By “end labeling” is meant that the non-radioactive label is stably attached, typically covalently bonded, to the 3′ end of the ribonucleic acid, i.e. to the 3′ terminal ribonucleotide of the ribonucleic acid. End-labeling according to the subject invention is accomplished by enzymatically attaching one or more, and in many cases a plurality of, non-radioactively labeled ribonucleotides to the 3′ end of the ribonucleic acid, such that at least one and often a stretch or domain of sequentially attached non-radioactively labeled ribonucleotides are present at the 3′ end of the end-labeled ribonucleic acid. By enzymatically attaching is meant that one or more non-radioactively labeled ribonucleotides are sequentially attached to the 3′ terminal ribonucleotide of the ribonucleic acid with an enzyme having polymerase activity.
A critical feature of the subject invention is that the polymerase activity is provided by a prokaryotic polymerase, more specifically a bacterial polymerase, where the polymerase exhibits poly(A) polymerase activity. By
Agilent Technolgies, Inc.
Chakrabarti Arun
Fredman Jeffrey
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