Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1993-05-27
2001-01-30
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06180338
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the analysis of deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), the determination of the presence of a predetermined specific DNA and/or RNA nucleotide sequence, and the exponential amplification of such a sequence.
BACKGROUND OF THE INVENTION
An ability to detect the presence of a nucleic acid molecule having a particular predetermined sequence is of substantial importance in a variety of fields, such as forensics, medicine, epidemiology and public health, and in the prediction and diagnosis of disease. Such an ability can aid criminal investigations, by excluding wrongly accused individuals or by implicating culpable parties. It can be exploited to permit the identification of the causal agent of infectious disease, or the characterization of tumors and tissue samples, or ensure the wholesomeness of blood products.
An ability to detect the presence of a particular nucleic acid sequence in a sample is important in predicting the likelihood that two individuals are related to one another, or that an individual will suffer from a genetic disease. Such an ability can also be used in assays to determine the purity of drinking water, milk, or other foods.
In many cases of interest, the desired nucleic acid sequence is present at a very low concentration in the sample. In such cases, unless assay sensitivity can be increased through the use of sophisticated labels, the presence of the desired molecule may escape detection. Assay sensitivity may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose: enzyme labels (Kourilsky et al.; U.S. Pat. No. 4,581,333); radioisotopic labels (Falkow et al., U.S. Pat. No. 4,358,535; Berninger, U.S. Pat. No. 4,446,237); fluorescent labels (Albarella et al., EP 144914); chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EP 119448), etc.
Although the use of highly detectable labeled reagents can improve the sensitivity of nucleic acid detection assays, the sensitivity of such assays remains limited by practical problems which are largely related to non-specific reactions that increase the background signal produced in the absence of the nucleic acid the assay is designed to detect. Thus, for some applications, the anticipated concentration of the desired nucleic acid molecule will be too low to permit its detection by any of the above-described methods.
One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid molecule whose detection is desired prior to performing the assay. In vivo recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized (Cohen et al., U.S. Pat. No. 4,237,224; Sambrook, J. et al., In:
Molecular Cloning: A Laboratory Manual
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment.
Recently, in vitro amplification methods have been developed. The impact of such methods has been phenomenal—without such amplification, most of the foregoing exemplary fields would not be possible. Thus, as the areas in which DNA amplification has expanded, the requirements placed upon various amplification techniques have changed. Accordingly, a very real and ongoing need exists for highly specific amplification techniques.
Perhaps the most widely practiced of these methods is the “polymerase chain reaction” (“PCR”) (Mullis, K. et al.,
Cold Spring Harbor Symp. Quant. Biol.
51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194), which references are incorporated herein by reference).
PCR achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified. Extension products incorporating the primers then become templates for subsequent replication steps. The method selectively increases the concentration of a desired nucleic acid molecule even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single or double stranded DNA.
The method involves the use of a DNA polymerase to direct the template-dependent, extension of a pair of oligonucleotide primers. The primer extension products then become templates for subsequent replication steps.
The precise nature of the two oligonucleotide primers of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5′→3′ linkage of the sugar-phosphate backbone of the molecule. Two DNA or RNA molecules may be linked together through the formation of a phosphodiester bond between the terminal 5′ phosphate group of one molecule and the terminal 3′ hydroxyl group of the second molecule. Polymerase dependent amplification of a nucleic acid molecule proceeds by the addition of a 5′ nucleoside triphosphate to the 3′ hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3′ terminus of a nucleic acid molecule. The oligonucleotide sequences of the two PCR primers are selected such that they contain sequences identical to, or complementary to, sequences which flank the sequence of the particular nucleic acid molecule whose amplification is desired. More specifically, the nucleotide sequence of the “first” primer is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3′ to the sequence of the desired nucleic acid molecule, whereas the nucleotide sequence of the “second” primer is selected such that it contains a nucleotide sequence identical to one present 5′ to the sequence of the desired nucleic acid molecule. Both primers possess the 3′ hydroxyl groups which are necessary for enzyme mediated nucleic acid synthesis.
The PCR reaction is capable of exponential amplification of specific nucleic acid sequences because the extension product of the “first” primer contains a sequence which is complementary to a sequence of the “second” primer, and thus will serve as a template for the production of an extension product of the “second” primer. Similarly, the extension product of the “second” primer, of necessity, contain a sequence which is complementary to a sequence of the “first” primer, and thus will serve as a template for the production of an extension product of the “first” primer. Thus, by permitting cycles of hybridization, polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved.
PCR technology is useful in that it can achieve the rapid and extensive amplification of a polynucleotide molecule (Mullis, K. B.,
Cold Spring Harbor Symp. Quant. Biol.
51:263-273 (1986); Saiki, R. K., et al.,
Bio/Technology
3:1008-1012 (1985); Mullis, K. B., et al.,
Met. Enzymol.
155:335-350 (1987), which references are incorporated herein by reference). Nevertheless, several practical problems exist with PCR. First extraneous sequences along the two templates can hybridize with the primers; this results in co-amplification due to such non-specific hybridization. As the level of amplification increases, the severity of such co-amplification also increases. Second, because of the ability of PCR to readily generate millions of copies for each initial template, accidental introduction of the end-product of
Auerbach Jeffrey I.
Beckman Coulter Inc.
Houtteman Scott W.
Kivinski Margaret A.
May William H.
LandOfFree
Method, reagent and kit for the detection and amplification... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method, reagent and kit for the detection and amplification..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method, reagent and kit for the detection and amplification... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2536647