Deducing Exon Connectivity by RNA-Templated DNA Ligation/Sequencing

• US 20150240232A1
• Filed: 01/22/2015
• Published: 08/27/2015
• Est. Priority Date: 10/19/2009
• Status: Active Application

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Zamore, Phillip D., Moore, Melissa J., Roy, Christian
• Application Number:
  US14/602,756
• Publication Number:
  US 20150240232A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2539/105 Involving introns, exons, or splice junctions

Abstract

A technology is described that is capable of generating high-throughput sequencing (HTS) read length DNA products to accurately and reliably provide exon connectivity information for alternatively spliced isoforms. The method is not limited by the initial size of the isoform as the technology removes the template oligonucleotide sequence and a newly formed full length ligated product provides an HTS-compatible read length sequence that comprises information that corresponds to the consecutive order of the exons in the original template oligonucleotide.

Claims

1. A composition comprising a ligamer comprising a plurality of complementarity regions, wherein at least two of said plurality of complementary regions are separated by a central region, wherein said central region has an exon-specific barcode sequence comprising an identifier code for a specific exon, wherein said at least two of said plurality of complementarity regions are complementary to the flanking regions of said specific exon.

2. The composition of claim 1, wherein each of said complementarity regions comprise a different nucleotide sequence.

3. The composition of claim 1, wherein said composition further comprises a template oligonucleotide comprising a plurality of hybridization sites.

4. The composition of claim 3, wherein said template oligonucleotide hybridization sites are complementary to said complementarity regions.

5. The composition of claim 3, wherein said template oligonucleotide comprises an mRNA oligonucleotide.

6. The composition of claim 5, wherein said mRNA oligonucleotide comprises said exon.

7. The composition of claim 6, wherein at least one of said hybridization sites comprises a flanking region of said exon.

8. The composition of claim 6, wherein said barcode sequence is non-complementary to said exon.

9. The composition of claim 1, wherein said ligamer comprises between approximately 5-5,000 nt.

10. The composition of claim 1, wherein said ligamer comprises between approximately 10-3,000 nt.

11. The composition of claim 1, wherein said ligamer comprises between approximately 20-1,000 nt.