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Universal-tagged oligonucleotide primers and methods of useRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidUniversal-tagged oligonucleotide primers and methods of use description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128654, Universal-tagged oligonucleotide primers and methods of use. Brief Patent Description - Full Patent Description - Patent Application Claims
REFERENCE TO RELATED APPLICATION [0001] The present application is a continuation of U.S. patent application Ser. No. 10/151,061, filed May 16, 2002, the entirety of which is incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to universal-tagged oligonucleotide primers, and to methods of using the primers for amplifying the genome. INTRODUCTION [0003] Whole genome amplification (WGA) is a valuable technique for amplification of the genome from minimal or limiting amounts of DNA for subsequent molecular genetic analysis. It is desirable that whole genome amplification is conducted to ensure that the amplification is not biased, meaning that all sequences in a sample should be amplified to the same extent. [0004] Whole genome amplification may be performed using either conventional or nonconventional PCR amplification methods. Conventional PCR entails the amplification and subsequent detection of specific DNA sequences which are precisely characterized in length and sequence using nondegenerate primers, while random, "non-conventional" PCR involves universal amplification of prevailing DNA or amplification of unknown intervening sequences which are not generally defined in length or sequence using degenerate primers. [0005] The use of specific primers to amplify genomic DNA (gDNA) is not practical or economical for multiplex applications, such as analyzing single-nucleotide polymorphisms (SNPs), while the use of random primers to amplify gDNA is typically not reproducible or predictable due to non-specific priming. SUMMARY OF THE INVENTION [0006] In one aspect, the invention provides a method for amplifying target DNA comprising multiple DNA sequences by polymerase chain reaction (PCR) by preparing a mixture of (1) the target DNA, (2) a set of single-stranded oligonucleotide primers, each primer comprising (i) a 3' specific region, (ii) a random region, and (iii) a 5' universal region, wherein the universal region serves as a first universal priming site (U1) for further PCR amplification with a complementary primer, (3) a DNA polymerase, and (4) multiple deoxynucleoside triphosphates (dNTPs) under conditions such that the PCR primers anneal to and prepare a copy of the multiple DNA sequences by primer extension of the target DNA, yielding an amplified product retaining the first universal priming site (U1). The target DNA amplified by this method may be, without limitation, gDNA or cDNA, and may include at least a fraction of the whole human genome. If desired, the amplified product retaining the U1 universal priming site may be further extended to encode a second universal priming site (U2), and further amplified with two primers complementing the first and second universal priming sites (U1 and U2). In one embodiment, the DNA polymerase for amplifying the target DNA is a Taq DNA polymerase, such as, for example, AMPLITAQ GOLD.RTM. DNA polymerase, AMPLITAQ GOLD.RTM. DNA polymerase, or the Stoffel fragment of AmpliTaq.TM. DNA polymerase. AMPLITAQ GOLD.RTM. is a DNA polymerase which is chemically modified to allow for a hot start during PCR. AMPLITAQ.RTM. is a DNA polymerase which is recombinantly produced and modified to remove exonuclease activity. If desired, the method of amplifying target DNA may include subjecting the amplified product to single-nucleotide polymorphism (SNP) genotyping. In a particular embodiment, the set of oligonucleotide primers used in the amplification reaction is designed based upon bioinformatic prediction of expected products, using in silico "e-PCR." [0007] In another aspect, the invention provides a universal-tagged single-stranded oligonucleotide primer for polymerase chain reaction (PCR), comprising, (1) a 3' specific region, (2) a random region, and (3) a 5' universal region. [0008] In yet another aspect, the invention provides a set of single-stranded oligonucleotide primers for amplification of genomic DNA (gDNA) in a polymerase chain reaction (PCR), each of the primers comprising (1) a 3' specific region, (2) a random region, and (3) a 5' universal region. [0009] In a further aspect, the invention provides a primer-target duplex that forms between the binding region of the single-stranded oligonucleotide primer and the target DNA. [0010] In all aspects of the invention, the 3' specific region of the oligonucleotide primers may, for example, be about 4 to 12 bases in length. In another embodiment, the 3' specific region may be designed to bind to a genomic sequence occurring in the human genome with a frequency of about 0.01% and 2.00%. In a further embodiment, the random region of the primers may be between about 2 and 15 bases in length. In primers with a random region of 4 bases in length, the sequence of the random region may include all 256 possible combinations of adenosine, cytosine, guanine, and thymidine. In a still further embodiment, the 5' universal region of the primers may be designed to have no significant homology to any segment in the human genome and further to be between about 12 and 35 bases in length. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 shows 5-mers that may serve as a specific region in a single-stranded oligonucleotide primer and their frequency of occurrence in the entire human genome. [0012] FIG. 2 is a schematic of PCR amplification of the target COX6b DNA sequence using two single-stranded oligonucleotide primers, each having a 3' specific region of 5 bases in length (5'-GCTCG), a random region of 4 bases in length (5'-NNNN), and a 5' universal region (U1 universal primer site or U2 universal primer site) to incorporate a universal U1 and a universal U2 priming site into a PCR-generated copy of the target COX6b DNA sequence. [0013] FIG. 3 is a schematic of the use of U1 and U2 primers to further amplify a PCR-generated copy of target DNA containing a universal U1 and universal U2 priming site. [0014] FIG. 4 is a schematic of PCR amplification using locked nucleic acid (LNA) substituted single-stranded oligonucleotide primers comprising a 3' specific region (5'-TTTtTtTtT) and a 5' universal region that may serve as a universal priming site for further PCR amplification. LNA residues are shown in capital letters, e.g. T, while normal nucleic acids are shown in lower case letters, e.g. t. [0015] FIG. 5 shows a region of the nucleotide sequence of the COX6 gene (SEQ ID NO: 35) with sequences underlined to indicate sites for annealing of primers and probes. The sequence of primers and probes that anneal to the COX6 gene are shown in capital letters directly above the target sequence. [0016] FIG. 6 shows the Tm calculation for the formation of duplexes between target DNA and primers either with or without a universal tag. [0017] FIG. 7 represents a schematic of the formation of a primer-target duplex between a universal-tagged primer and the target DNA. Vertical solid lines are used to denote complete hydrogen bonding between bases in the specific region of the primer and the target while vertical hatched lines are used to denote less than complete hydrogen bonding between all the bases in the random region of the primer and the target. DETAILED DESCRIPTION [0018] A. Definitions Continue reading about Universal-tagged oligonucleotide primers and methods of use... Full patent description for Universal-tagged oligonucleotide primers and methods of use Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Universal-tagged oligonucleotide primers and methods of use patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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