Genetic mutational analysis
AssigneesBioSkryb Genomics, Inc., St. Jude Children's Research Hospital
InventorsJay A.A. WEST, Charles GAWAD, Sivaraman NATARAJAN, Robert CARTER, Veronica GONZALES-PENA
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Abstract
Provided herein are compositions and methods for accurate and scalable Primary Template-Directed Amplification (PTA) nucleic acid amplification and sequencing methods, and their applications for mutational analysis in research, diagnostics, and treatment. Such methods and compositions facilitate highly accurate amplification of target (or “template”) nucleic acids, which increases accuracy and sensitivity of downstream applications, such as Next-Generation Sequencing.
Claims48
A method of determining a mutation comprising:
- a. exposing a population of cells to a gene editing method, wherein the gene editing method utilizes reagents configured to effect a mutation in a target sequence;
- b. isolating single cells from the population;
- c. providing a cell lysate from a single cell;
- d. contacting the cell lysate with at least one amplification primer, at least one nucleic acid polymerase, and a mixture of nucleotides, wherein the mixture of nucleotides comprises at least one terminator nucleotide which terminates nucleic acid replication by the polymerase, and
- e. amplifying the target nucleic acid molecule to generate a plurality of terminated amplification products, wherein the replication proceeds by strand displacement replication;
- f. ligating the molecules obtained in step (e) to adaptors, thereby generating a library of amplification products;
- g. sequencing the library of amplification products, and
- h. comparing the sequences of amplification products to at least one reference sequence to identify at least one mutation.
Dependencies are inferred from the claim text.