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US 11,015,213 B2Granted
Method of preparing cell free nucleic acid molecules by in situ amplification
AssigneeCirculogene Theranostics, LLC
InventorChen-Hsiung Yeh
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Abstract
Methods for in situ amplification (ISA) of cfNA, such as cfDNA, in a sample are provided wherein the cfNA in the sample is not subject to a nucleic acid purification step. The methods disclosed may be used to generate an analyzable pool of cfNA present in the sample. The analyzable pool may be used with a variety of analytical techniques to characterize the nucleic acid in the sample. Methods of diagnosis, determining a therapeutic intervention and monitoring of a subject are also provided.
Claims35
A method of in situ amplification of a cell-free nucleic acid (cfNA) the method comprising the steps of:
- a providing a liquid sample containing a plurality of cfNA;
- b performing at least one processing step on the sample;
- c subjecting the sample to a sequential heating program and converting at least a portion of the cfNA in the sample to a modified cfNA using an enzyme mixture to add an exogenous nucleic acid sequence to at least one of the 5′ or 3′ ends of at least a portion of the cfNA in the sample to create an amplifiable cfNA pool, wherein the exogenous nucleic acid sequence contains a primer site capable of binding a primer and the exogenous nucleic acid sequence has a degenerate nucleic acid sequence flanking at least one side of the primer site;
- and d amplifying the amplifiable cfNA pool to produce an analyzable pool of cfNA wherein the cfNA in the sample is not subject to a nucleic acid purification step.
1 more element
Dependencies are inferred from the claim text.