Sample preparation methods, systems and compositions
Inventors
Blauwkamp, Timothy A. • Sit, Rene • Vilfan, Igor D.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11180800-B2
Publication Date
2021-11-23
Expiration Date
Abstract
The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.
Core Innovation
The invention provides methods for performing an amplification reaction or primer extension on a sample that includes a mixture of first RNA and cell-free DNA that does not comprise a sequence complementary to the first RNA. The workflow tags the cell-free DNA with a first tag without using a transposase, and tags the first RNA with a second tag. After tagging, amplification or primer extension is performed on the first DNA using a polymerase that is selective for DNA templates, and a reverse transcriptase synthesizes a complementary DNA (cDNA) strand from the first RNA.
A related aspect enables concurrent processing of different nucleic acid forms in a sample that comprises an RNA molecule and a DNA molecule. The method denatures the different nucleic acid forms, attaches a first adapter to one end of the RNA molecule and a second adapter to one end of the DNA molecule where the DNA molecule is cell-free DNA, and generates an RNA molecule attached to a first adapter and a DNA molecule attached to a second adapter. Primers are hybridized to the adapters, and extension reactions amplify the RNA molecule attached to the first adapter and amplify the DNA molecule attached to the second adapter to generate an RNA extension product and a DNA extension product.
The concurrent processing continues by attaching third and fourth adapters comprising priming elements to the RNA extension product and the DNA extension product, respectively, where the third and fourth adapters comprise identical or different sequences. The method amplifies the RNA extension product attached to the third adapter and the DNA extension product attached to the fourth. The document context also describes distinguishing nucleic-acid structural forms and concurrent detection of different nucleic-acid forms using particle-protected nucleic acids such as exosomes.
Claims Coverage
The partial set includes two independent claims, each covering multiple inventive features for handling and amplifying RNA together with cell-free DNA.
Tagging RNA and cell-free DNA in non-complementary mixtures
Providing a sample comprising a mixture of first DNA and first RNA where the first DNA is cell-free DNA and does not comprise a sequence complementary to the first RNA; tagging the first DNA with a first tag without using a transposase; and tagging the first RNA with a second tag.
DNA-template-selective amplification and cDNA synthesis
Performing an amplification or primer extension reaction on the first DNA with a polymerase that is selective for DNA templates; and synthesizing a complementary DNA (cDNA) strand from the first RNA with a reverse transcriptase.
Concurrent processing of RNA and cell-free DNA via adapters and extension
Denaturing different nucleic acid forms in a sample comprising an RNA molecule and a DNA molecule where the DNA molecule is cell-free DNA; attaching a first adapter to one end of the RNA molecule and attaching a second adapter to one end of the DNA molecule to generate adapter-attached RNA and adapter-attached DNA; hybridizing primers to the first and second adapters and performing extension reactions to generate an RNA extension product and a DNA extension product; attaching third and fourth adapters comprising priming elements to the RNA extension product and the DNA extension product; and amplifying the RNA extension product attached to the third adapter and the DNA extension product attached to the fourth adapter.
Overall, the independent claims center on tagging RNA and cell-free DNA in a mixed sample that is non-complementary, then using a DNA-template-selective polymerase together with reverse transcription to produce cDNA from RNA, and on adapter-mediated concurrent processing of RNA and DNA followed by further amplification.
Stated Advantages
Enables performing amplification or primer extension on a sample comprising first RNA and cell-free DNA that is not complementary to the first RNA.
Supports concurrent processing of different nucleic acid forms comprising RNA and cell-free DNA in a single workflow.
Allows amplification of RNA attached to adapter priming elements and amplification of DNA attached to adapter priming elements using extension reactions.
Enables distinguishing sequences originating from first DNA versus first RNA using identifying sequences on adapters or tags.
Documented Applications
Cancer detection by detecting tumor genes, mutations, and gene biomarkers using nucleic-acid detection methods described in the document context.
Fetal health assessment including IVF embryo genetic and infection-risk assessment and detection related to chromosomal aberrations and conditions such as Down syndrome, Trisomy 18, Trisomy 13, sex chromosome aneuploidy, and Prader-Willi syndrome.
Infection detection by detecting pathogens using both RNA and DNA from samples, including latent versus active infection markers, and using particle-protected nucleic acids such as exosomes.
Concurrent detection use cases for particle-protected nucleic acids and diverse pathogen classes using nucleic-acid form discrimination.
Interested in licensing this patent?