Icahn School of Medicine at Mount Sinai

Mount Sinai School of Medicine

Anitha Jayaprakash

Ravi Sachidanandam

Brian Brown

Andrew Chess

The present disclosure relates to methods for determining recombination diversity at a genomic locus of interest. The method includes fragmenting nucleic acids isolated from immune cells, ligating adaptors to the fragmented or amplified nucleic acids, and selectively amplifying nucleic acids containing a recombined junction at the genomic locus of interest. Selective amplification is achieved by using a first primer that hybridizes to an adaptor sequence and a second primer that hybridizes at a constant region downstream of the recombined junction. The selectively amplified nucleic acids may be sequences and analyzed to determine recombination diversity at the genomic locus.

Methods for determining recombination diversity at a genomic locus

Modified nucleic acid adapters are provided that collectively provide a mixture of nucleotides at the 3' end of 5&#x2032; adapters and at the 5&#x2032; end of 3&#x2032; adapters such that at least one adapter in each set has any given nucleotide at position 1, i.e., the nucleotide position available for ligation to a small RNA, and has any given nucleotide at position 2 adjacent to position 1 for use in overcoming bias during nucleic acid manipulation, such as small RNA characterization and/or profiling by, e.g., deep sequencing, along with methods for use of the modified adapters in small RNA characterization. The modified adapters have at least two mixed nucleotides at the adapter terminus to be ligated to a nucleic acid such as a small RNA.

Pooled adapter strategy for reducing bias in small RNA characterization

A method for preparing circular double stranded mitochondrial DNA (mtDNA) substantially free of genomic DNA (gDNA) comprising the steps of: providing a cellular lysate free of protein and RNA contaminants, precipitating cellular debris and proteins out of said lysate and obtaining a solution comprising purified circular double stranded mitochondrial DNA (mtDNA) and genomic DNA (gDNA), incubating said solution with an amount of Hind Exonuclease V for a time and at a temperature effective to cleave non-circular DNA and obtain circular double stranded mtDNA, incubating said circular double stranded mtDNA with an amount of Ampure beads effective to bind said circular double stranded mtDNA, washing said beads with ethanol, and eluting said mtDNA from said beads, wherein said method is free of ultra-centrifugation.

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New York, NY, United States of America

Anitha Jayaprakash