Ligase-assisted nucleic acid circularization and amplification
AssigneeGeneral Electric Company
InventorsRyan Charles Heller, John Richard Nelson, Erik Leeming Kvam
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Abstract
Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.
Claims34
A method for generating a single-stranded DNA circle from a linear DNA, the method comprising:
- (a) providing the linear DNA;
- (b) incubating the linear DNA with a polynucleotide kinase in presence of a phosphate donor to generate a ligatable DNA sequence having a phosphate group at a 5′ terminal end and a hydroxyl group at a 3′ terminal end;
- and
- (c) incubating the ligatable DNA sequence with a ligase that is capable of template-independent, intra-molecular ligation of a single-stranded DNA sequence to generate the single-stranded DNA circle,
- wherein all the steps of the method are performed in a single reaction vessel without any intervening isolation or purification steps.
A method of generating a single-stranded DNA circle from a linear DNA, the method comprising:
- (a) providing the linear DNA;
- (b) optionally incubating the linear DNA with a polynucleotide kinase in presence of adenosine triphosphate to generate a ligatable DNA sequence having a phosphate group at a 5′ terminal end and a hydroxyl group at a 3′ terminal end;
- (c) incubating the linear DNA sequence or the ligatable DNA sequence with an adenylating enzyme in presence of adenosine triphosphate to generate a 5′ adenylated DNA sequence;
- and
- (d) incubating the 5′ adenylated DNA sequence with a non-adenylated ligase, which is capable of template-independent intra-molecular ligation of a 5′ adenylated, single-stranded DNA sequence to generate the single-stranded DNA circle,
- wherein all the steps of the method are performed in single reaction vessel without any intervening isolation or purification steps.
Dependencies are inferred from the claim text.