Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-29
2001-08-21
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06277578
ABSTRACT:
TECHNICAL FIELD
The invention relates to nucleic acid detection. More specifically, the invention relates to detection of amplified, targeted, predetermined nucleic acid sequences in nucleic acid target/probe hybrids, and the various applications of target nucleic acid amplification.
BACKGROUND OF THE INVENTION
Several methods of amplification coupled with detection of the presence or absence of a nucleic acid target sequence are currently used in the art. The amplification of a nucleic acid target that is present at a low concentration within a nucleic acid sample is useful for enhancing the sensitivity of a detection method. Known amplification methods include, inter alia, polymerase chain reaction, ligase chain reaction, repair chain reaction, amplification of transcripts, self-sustained sequence replication (3SR), ligation activated transcription (LAT), strand displacement amplification (SDA) and rolling circle replication. These processes are discussed in greater detail in the “Detailed Description of the Invention”.
There are several ways to use nucleic acid amplification methods in a nucleic acid detection method. One approach is to make the amplification primers complementary to the nucleic acid target. Then the presence of an amplification product is indicative of the presence of the nucleic acid target. In another approach, amplification primers are used to amplify a region of nucleic acid that contains a nucleic acid target. Then, another method is used to detect the presence of the nucleic acid target sequence within the amplification product.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
A traditional type of process for the detection of hybridized nucleic acid uses labeled nucleic acid probes to hybridize to a nucleic acid sample. For example, in a Southern blot technique, a nucleic acid sample is separated in an agarose gel based on size and affixed to a membrane, denatured, and exposed to the labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane. Probes used in Southern blots have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and acridinium esters.
Another type of process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. PCR-based methods are of limited use for the detection of nucleic acid of unknown sequence.
In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g. incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
Fluorescence techniques are also known for the detection of nucleic acid hybrids. U.S. Pat. No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes that are fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base variances in sequences. Some fluorescence techniques involve digestion of a nucleic acid hybrid in a 5′ to 3′ direction to release a fluorescent signal from proximity to a fluorescence quencher, for example, TaqMan® (Perkin Elmer; U.S. Pat. Nos. 5,691,146 and 5,876,930).
Enzymes having template-specific polymerase activity for which some 3′ to 5′ depolymerization activity has been reported include
E. coli
DNA Polymerase (Deutscher and Kornberg,
J. Biol. Chem.,
244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al.,
Biochemistry
30:526-37 (1991); Tabor and Richardson,
J. Biol. Chem.
265: 8322-28 (1990)),
E. coli
RNA polymerase (Rozovskaya et al.,
Biochem. J.
224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak,
J. Biol. Chem.
255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al.,
J. Biol. Chem.
269:24195-202 (1994)). A template-dependent polymerase for which 3′ to 5′ exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA polymerase (de Vega, M. et al.
EMBO J.,
15:1182-1192, 1996).
A variety of methodologies currently exist for the detection of single nucleotide polymorphisms (SNPs) that are present in genomic DNA. SNPs are DNA point mutations or insertions/deletions that are present at measurable frequencies in the population. SNPs are the most common variations in the genome. SNPs occur at defined positions within genomes and can be used for gene mapping, defining population structure, and performing functional studies. SNPs are useful as markers because many known genetic diseases are caused by point mutations and insertions/deletions.
In rare cases where a SNP alters a fortuitous restriction enzyme recognition sequence, differential sensitivity of the amplified DNA to cleavage can be used for SNP detection. This technique requires that an appropriate restriction enzyme site be present or introduced in the appropriate sequence context for differential recognition by the restriction endonuclease. After amplification, the products are cleaved by the appropriate restriction endonuclease and products are analyzed by gel electrophoresis and subsequent staining. The throughput of analysis by this technique is limited because samples require processing, gel analysis, and significant interpretation of data before SNPs can be accurately determined.
Single strand conformational polymorphism (SSCP) is a second technique that can detect SNPs present in an amplified DNA segment (Hayashi, K.
Genetic Analysis: Techniques and Applications
9:73-79, 1992). In this method, the double stranded amplified product is denatured and then both strands are allowed to reanneal during electrophoresis in non-denaturing polyacrylamide gels. The separated strands assume a specific folded conformation based on intramolecular base pairing. The electrophoretic properties of each strand are dependent on the folded conformation. The presence of single nucleotide changes in the sequence can cause a detectable change in the conformation and electrophoretic migration of an amplified sample relative to wild type samples, allowing SNPs to be identified. In addition to the limited throughput possible by gel-based techniques, the design and interpretation of SSCP based experiments can be difficult. Multiplex analysis of several samples in the same SSCP reaction is extremely challenging. The sensitivity required in mutation detection and analysis has led most investigators to use radioactively labeled PCR products for this technique.
In a process to amplify and detect single nucleotide polym
Andrews Christine Ann
Hartnett James Robert
Leippe Donna
Lewis Martin K.
Mandrekar Michelle
Horlick Kenneth R.
Promega Corporation
Strzelecka Teresa
Welsh & Katz Ltd.
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