Methods and compositions for enrichment of target nucleic acids
AssigneeChapter Diagnostics, Inc.
InventorsChunlin Wang, Zhihai Ma, Baback Gharizadeh
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Abstract
The present disclosure relates to a method of target enrichment and amplification of selective target regions of genomes or nucleic acid samples and further analysis by systems such as NGS. The disclosure provides methods and kits that can be used in numerous downstream procedures including DNA sequencing. The disclosed methods can be utilized to sequence the target nucleic acid sequences such as to detect the presence of genetic variations for biological assays, assessment of disease, to count copies of target regions, and to allow such target enrichment prior to sequencing.
Claims15
A method for enrichment of target nucleic acid sequences, comprising the steps of:
- directionally ligating partially double-stranded blocking adapters to the ends of one or more nucleic acid samples;
- combining a portion of the ligated nucleic acid samples with a biotin-labeled common primer A and one or more forward strand-specific primers in one test reaction, wherein the forward strand-specific primers comprise universal sequence B;
- combining a second portion of the ligated nucleic acid samples with a biotin-labeled common primer A and:
- one or more reverse strand-specific primers in a second test reaction, wherein the reverse strand-specific primers comprise universal sequence B;
- subjecting each test reaction to amplification to generate a first set of amplicons;
- enriching the first set of amplicons by pooling the first set of amplicons together and then binding the pooled first set of amplicons to streptavidin-coated beads;
- and
- subjecting a portion of the first set of amplicons to a second round of amplification using common primer A and common primer B to generate a final set of amplicons.
A kit comprising:
- at least one partially double-stranded blocking adapter, wherein strand L of the at least one partially double-stranded blocking adapter comprises a barcode and a universal sequence A;
- at least one forward strand-specific primer;
- at least one reverse strand-specific primer;
- a biotin-labeled common primer A;
- and
- common primer A and common primer B;
- wherein the at least one forward strand-specific primer and at least one reverse strand-specific primer each comprise universal sequence B.
Dependencies are inferred from the claim text.