10 results about "Multiple displacement amplification" patented technology

The present disclosure provides primers, primer sets, kits and methods for multiple displacement amplification, especially in combination with nucleic acid sequencing. The primers comprise self-complementary sequences at their 5′ termini and random or semi-random sequences at their 3′ termini. Use of such primers facilitates handling of multiple samples, increases sequence coverage uniformity, and improves sequencing error corrections.

The application discloses a mutant PWT polymerase and a preparation method and application thereof, and relates to the technical field of biology. The mutant PWT polymerase has any one of A1) to A2): A1), an amino acid sequence shown in amino acids 407 to 986 of SEQ ID NO. 1; A2), an amino acid sequence obtained by connecting a tag to the N terminal or / and C terminal of the amino acid sequence shown in A1). The mutant PWT polymerase can obviously perform isothermal amplification under room temperature conditions and can tolerate a higher salt concentration, and is expected to become an ideal tool for rolling circle amplification and multiple displacement amplification.

A method of selective DNA amplification of a DNA mixture comprising a first population of DNA and a second population of DNA, wherein:—a) a catalytically dead Staphylococcus aureus Cas9 complex (dSaCas9) comprising a dSaCas9 protein or derivative thereof complexed with one or more guide RNAs having selective binding affinity for DNA sequences preferentially present in the first population of DNA compared to the second population of DNA is contacted with the DNA mixture under a first reaction condition, said first reaction condition being suitable for binding of the dSaCas9 complex to DNA sequences for which it has a binding affinity, and thenb) a strand-displacing DNA polymerase is contacted with the DNA mixture under a second reaction condition, said second reaction condition being suitable for amplification being suitable for amplification activity of the strand-displacing DNA polymerase; and related kits and uses.

Disclosed herein are mutants of bacteriophage Phi29 DNA polymerase with improved primer recognition, compared to the wild-type enzyme. Certain mutants comprise one or both of the mutations K64R or M97K. The provided mutants are capable of using more efficiently shorter and longer random synthetic DNA primers than wild-type Phi29 DNA polymerase, generating more amplification product in Multiple Displacement Amplification (MDA) reactions. The inventive mutants amplify human genomic DNA with less bias and better coverage in comparison to reactions carried out with wild-type Phi29 DNA polymerase.

The present disclosure provides primers, primer sets, kits and methods for multiple displacement amplification, especially in combination with nucleic acid sequencing. The primers comprise self-complementary sequences at their 5′ termini and random or semi-random sequences at their 3′ termini. Use of such primers facilitates handling of multiple samples, increases sequence coverage uniformity, and improves sequencing error corrections.

One aspect relates to a nucleic acid amplification method that comprises the step of amplifying nucleic acids by adding a first primer, a second primer and a nucleic acid polymerase to a sample containing a target nucleic acid. According to the nucleic acid amplification method according to one aspect, by performing parallel multiple displacement amplification using the first primer containing the barcode sequence, the increased parallelism and throughput of MDA dramatically reduces the time and cost for NGS library production, the barcode bias phenomenon can be resolved by adjusting the ratio at which the first primer is added, and the quality of amplification can also be improved using a laser-based cell separation method. Therefore, the nucleic acid amplification method can be usefully used for large-scale single-cell whole genome analysis.

Disclosed herein are mutants of bacteriophage Phi29 DNA polymerase that have improved primer recognition compared to the wild-type enzyme. Certain mutants contain one or both of the K64R or M97K mutations. The provided mutants are capable of using shorter and longer random synthetic DNA primers more efficiently than the wild-type Phi29 DNA polymerase, producing more amplification products in multiple displacement amplification (MDA) reactions. Compared to reactions performed with the wild-type Phi29 DNA polymerase, the mutants of the present invention amplify human genomic DNA polymerase with less deviation and higher coverage.

The invention discloses a high-fidelity single cell whole genome amplified polymerase mutant and a kit and application thereof. The polymerase mutant is a Phi29 DNA polymerase mutant which is any one of M1, M2, M3 and M4, and the amino acid sequences of the polymerase mutant are shown as SEQ ID NO.2-5 respectively. The Phi29 DNA polymerase mutant disclosed by the invention contains four mutation sites and can be stored for a long time under a room temperature condition. Compared with a wild type, the Phi29 DNA polymerase mutant M4 disclosed by the invention has excellent thermal stability. Under the reaction condition of 60 DEG C, 90% or above of enzyme activity can still be kept after 6 hours. The mutant shows higher amplification uniformity, reaction rate and fidelity in DNA amplification reaction, can be used for catalyzing multiple replacement amplification and rolling circle replication reaction, and can be used for preparing a PGT detection kit.

The application relates to the technical field of molecular biology, and discloses a mutant DNA polymerase as well as a preparation method and application thereof, wherein the amino acid sequence of the mutant DNA polymerase is shown as SEQ ID NO. 4, the amino acid sequence is obtained by mutation on the basis of a wild-type FWT polymerase, and the mutation sites include that alanine at the 80th position is mutated into arginine, asparagine at the 107th position is mutated into aspartic acid, glutamic acid at the 172nd position is mutated into arginine, asparagine at the 325th position is mutated into aspartic acid, aspartic acid at the 423rd position is mutated into alanine, serine at the 490th position is mutated into alanine, and phenylalanine at the 491st position is mutated into alanine. The mutant DNA polymerase has better continuous synthesis, strand displacement and high-salt resistance than the wild type under room temperature conditions, and is an ideal tool for rolling circle amplification and multiple displacement amplification.