181 results about "Primer dimer" patented technology

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention describes composition and methods of use for novel hairpin blocked-cleavable primers. In one embodiment unblocking occurs through action of RNase H2. The method improves the specificity of PCR and reduces primer dimer events, enabling higher level multiplex reactions. Additionally, the invention protects RNA-containing primers from attack by single-strand RNases.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Methods are provided for amplification of a nucleic acid sequence. The method use RNA/DNA chimeric oligonucleotides as primers. The primers have RNA residues scattered along their length and no two ribonucleotides in the prime are adjacent to one another. The methods are useful for reducing non-specific amplification products, such as primer dimers. The invention also provides kits comprising RNA/DNA chimeric oligonucleotide primers for practicing the amplification methods.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Methods and kits for efficient amplification of nucleic acids are provided. The methods comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having terminal mismatch primer-dimer structure. The methods also comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having nucleotide analogues. The methods enhance efficiency of nucleic acid amplification reaction by reducing non-specific amplification reactions.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The present invention provides modified primers for use in the amplification of a nucleic acid sequence. Amplifications carried out using the modified primers result in less template-independent non-specific product (primer dimer) compared to amplifications carried out using unmodified primers.

The invention provides a real-time fluorescence quantitative PCR (Polymerase Chain Reaction) kit for detecting the expression level of HER2 genes and relates to a PCR kit. The real-time fluorescence quantitative PCR kit comprises PCR reaction liquid of the HER2 genes, PCR reaction liquid of ACTB genes, a Taq enzyme system, a control product and a packaging object; and the adopted primer is an annular primer, 3-8 self-designed basic groups are at the 5' end, and can be combined with the sequence at the 3' end under proper conditions so as to form double chains. The real-time fluorescence quantitative PCR kit has the advantages that the non-specific amplification products especially primer dimer generated by nonspecific amplification products especially the primer can be effectively prevented from being formed, the specificity is increased; and a relative-quantitative RT-PCR method is utilized for detecting the expression level of the HER2 genes of a patient with the breast cancer, adopts a 2-delta deltaCt value method for quantifying the detected result, and can be used for diagnosis of early stage and transferring of the breast cancer and assisting multiple fields such as clinicalmedicine selection and prognosis.

The invention discloses a construction method of a ctDNA ultra-low-frequency mutation detection library, a kit and an analysis method of library detection data. The construction method comprises steps as follows: S1, cfDNA is extracted from whole blood; S2, the terminal of cfDNA is restored and an A basic group is added to the 3'terminal; S3, the terminal of the cfDNA obtained in S2 is connected with connectors containing random label sequences; S4, primers for multiplex PCR (polymerase chain reaction) are designed according to the sequences of the connectors and an object region for object region capturing; S5, a PCR product in S4 is subjected to magnetic bead purification, and small-fragment DNA not subjected to non-specific amplification and primer dimer are removed; S6, a product in S5 is subjected to PCR amplification, an index sequence is introduced, and the ctDNA ultra-low-frequency mutation library is obtained. According to the method, PCR mistakes and sequencing mistakes are eliminated, and the detection specificity is improved.

The invention relates to a primer middle sequence interference PCR (Polymerase Chain Reaction) technology. The improved PCR technology is characterized in that one segment of relatively non-complemented or same-sequence basic group primer molecules in the intermediate domain of primers perform the antisense interference inside and outside so as to competitively destroy the polymerization among the primers to selectively inhibit the primer dimer (PD) from being amplified. For the interference of the intermediate domain of the primers, based on the primers optimally selected by the conventional design principle, the technology that the intermediate domain (ID) of a pair of the primers are in parallel but are not complemented with each other or are in the same sequence or/and the technology that ID antisense oligonucleotides (Oligo) are added into the primers to perform the interference action or/and the Oligo antonymy is carried out in the primer molecules via the ID so as to perform the interference action are adopted, or the combined technology of the three types of the technologies is adopted. As a result, only the ID of the primers is interfered while the target specific amplification is not influenced; the combining force acting on the minority of base-group pairing hydrogen bonds at the tail end and the base-group hydrogen bonds outside the tail end due to the action of the primers is dispersed to a maximum extent, so that the PD is selectively inhibited. Therefore, the PD accumulation in the PCR system is avoided. If the mineral oil is additionally used, the sealed primers can slowly release the hot starting and the UDG pretreatment so as to prevent aerosol glue as a byproduct of the PCR system from causing the pollution. Consequently, the nucleic acid is amplified reliably and the real-time fluorescence PCR is quantified accurately.

The invention discloses a low-frequency gene fusion detection method and a low-frequency gene fusion detection device. The detection method comprises the following steps: S1, extracting cfDNA from whole blood; S2, performing tail end repairing on the cfDNA and adding A basic group at the 3' end; S3, connecting a joint containing a random label sequence; S4, designing a multiplex-PCR primer and performing target area capture; S5, performing magnetic bead purification on the PCR product in the S4, removing small fragment DNA not subjected to non-specific amplification and a primer dimer, and introducing an index sequence to obtain a ctDNA ultralow-frequency mutation library; S7, performing computer sequencing; S8, performing clustering analysis on data after library sequencing through the random label sequence; and S9, entering variation detection analysis after the quality control of the remaining data is qualified. By application of the technical scheme of the invention, the low-frequency mutation detection sensitivity is improved.

Methods and kits for efficient amplification of nucleic acids are provided. The disclosure generally relates to methods and kits for nucleic acid amplification of target nucleic acids of interest. The methods described herein promote the synthesis of the target nucleic acid (i.e., template nucleic acid) by reducing the production of undesirable primer-dimer structures and chimeric nucleic acid products during the amplification process by using novel modified primers.

The invention discloses a construction method, a kit and application of a plasma free DNA methylation detection library. The construction method comprises the following steps: S1 of extracting plasmafree DNA from a blood sample; S2 of performing tail end repair and 3' terminal A base addition on the plasma free DNA; S3 of connecting the tail ends of the plasma free DNA obtained in the S2 with C methylation modified connectors; S4 of performing bisulfite conversion on a product obtained in S3 and performing PCR expansion; S5 of performing magnetic bead purification on the PCR product of S4 andremoving small-fragment DNA which is not amplified in a non-specific mode and primer dimer; S6 of performing PCR amplification on a product of S5 and performing magnetic bead purification to obtain aplasma free DNA methylation detection library. By means of the technical scheme of the construction method disclosed by the invention, an initial amount of general microscale methylation library construction is reduced, library construction steps are simplified, library recycling efficiency is improved, and a foundation is established for sequentially screening tumor diagnosis markers through plasma free DNA methylation difference locus.

The invention discloses application of graphene oxide to polymerase chain reaction (PCR). The application is implemented according to the method that the PCR is carried out under the action of the graphene oxide. The invention finds that the graphene oxide has the advantages of obviously increasing the specificity and the sensitivity of PCR and amplifying the yield by being applied to the PCR technology for the first time and can eliminate a primer dimer formed in amplification, in addition, the graphene oxide has a wide optimization interval and can be widely applied to DNA templates of various concentrations and complex degrees. Compared with other carbon nanomaterials applied to the PCR technology, the graphene oxide has a more excellent comprehensive effect on optimizing the PCR. In addition, the graphene oxide is low in cost, mature in preparation process, and simple to operate when applied to the PCR technology.

The invention relates to a method for performing multiplex PCR by utilizing hairpin primers and a kit as well as application thereof to the establishment of a second-generation sequencing platform library. A specific sequence at the 3'end of each of the hairpin primers is completely paired with a target segment, the melting temperature is 80 DEG C or higher, and a hairpin structure is only opened in the degeneration process when the PCR is performed. The non-specific amplification and the primer dimer formation can be effectively reduced, so that the uniformity of multiple PCR products is improved. The method and the kit as well as the application thereof provided by the invention have the benefits that the two rounds of reaction is performed by utilizing the multiple hairpin primers, so that the second-generation sequencing library is quickly and conveniently established; different templates are distinguished through joint primers carrying different bar code sequences, and the different joint primers carry a same universal amplification primer, so that the difference among different template amplification products is reduced.

The present invention relates to two visual detection reagents of isothermal amplification products of nucleic acids, the two visual detection reagents are that 1-(1-hydroxy-2-naphthalene azo)-6-nitro-2-naphthol-4-sodium sulfonate or derivatives thereof (hereinafter referred to as the reagent 1) and 4-(2-pyridine azo) resorcinol sodium salt or derivatives thereof (hereinafter referred to as the reagent 2), the two visual detection reagents are used alone, at the beginning of a reaction, the reagent 1 combines with magnesium ions to become red, and the reagent 2 combines with the magnesium ions to form an orange complex, with the reaction, by-product pyrophosphate groups combines the magnesium ions to form a precipitation, the reagent 1 or loses the magnesium ions to become blue from red, the reagent 2 becomes yellow from orange, amplification reaction results can be judged by color change, detection without opening of a cover can be realized by the method, the aerosol pollution can be overcome, and the false positive problem caused by primer dimmers can be solved.

Methods, compositions, systems and kits to amplify or improve amplification of target-specific amplification products by reducing non-specific amplification products (e.g., primer-dimers) when amplifying multiple different nucleotide regions. The methods, compositions, systems and kits described herein may include, or include the use of, one or more resolvases that recognize and bind to and/or cut an aberrant DNA structure.

The invention discloses a primer for amplifying a short-chain RNA (ribonucleic acid) and a related method thereof. The primer is oligonucleotide; a fragment of nucleotide sequence at the 5' end of the primer is fixed, and forms a structure with a nucleotide loop and a nucleotide stem; the 3' end of the primer is connected with 6 to 8 nucleotides, and is paired with the 3' end of a mature miR to form specific complementary binding; the 3' end of the nucleotide loop contains a fragment of nucleotide sequence with GC content of over 70 percent, and the fragment of nucleotide sequence is called a universal probe region; nucleotides on the 8th to 30th sites at the 5' end of the primer form a universal reverse primer region. The primer has an internal double-chain structure, and cannot be bound to a specific sequence in a nucleotide chain under the action of steric hindrance, and the reverse transcription of the sequence is avoided; the primer is only specifically paired with and bound to the 3' end for specific reverse transcription. The primer is high in specificity, easy to design, convenient to synthesize and suitable for the reverse transcription of the short-chain RNA, especially the mature miR, and the formation of a primer dimer is avoided.

The invention relates to a multiple PCR primer used for simultaneously detecting infectious Bovine Rhinotracheitis virus and akabane virus as well as its design method, and relates to detection of infectious Bovine Rhinotracheitis virus and akabane virus. The size of a PCR amplification fragment corresponded to an infectious Bovine Rhinotracheitis virus primer in the multiple PCR primer is 311bp, the size of the PCR amplification fragment corresponded to akabane virus id 392bp. The method comprises the following steps: 1) designing multiple PCR primer combination; 2) screening the primer in the primer combination, keeping the primer which can not synthesize a primer dimer; 3) determining the competition advantage and disadvantage states of the kept primer, comparing GC% and base number in the kept primer, selecting the kept primer with high GC content and determining as the primer with excellent competition state, performing a step 5); otherwise, performing a step 4); 4) selecting the primer with poor competition state again, wherein the concrete step repeats the step 2) and determining the kept primer according to the step 3) again; and 5) performing amplification on the primer with the excellent competition state.