Sigma-Aldrich Co. LLC

Brian W Ward

Thomas F Bittick

David M Ornitz

Carol A Kreader

Keming Song

Michael G Deines

Stephanie Uder

Ernest J Mueller

Jessica Copeland

Deborah Vassar

Jaime K Robert

Chuan Li

Fuqiang Chen

Kenneth E Heuermann

Ron Hernan

Timothy Seebeck

Leanne M Snyder

Kristen E Opper

Michael R Deines

Kenneth B Heuermann

Kristen E Bettinger

Methods are provided herein for identifying rare and/or unknown DNA sequences by next-generation sequencing approaches. Isolated double-stranded (ds), single-stranded (ss), or ds/ss DNA is fragmented and the fragments are polished, phosphorylated, and tailed, as necessary. Fragmentation can be enzymatic or mechanical. A universal adapter sequence is ligated to each fragment, wherein the adapter can have a top strand without a 5' phosphate, a 3&#x2032; with an &#x2014;H in place of the &#x2014;OH, and/or a 3&#x2032; extra base complementary to any base added to the polished fragments. The ligatamers may then serve as templates for amplification using a forward primer complementary to the adapter sequence and a reverse primer targeted to the fragment sequence. Compositions produced by these methods and kits adapted for performing these methods are also described herein.

Methods for next generation genome walking and related compositions and kits

Methods for primer switching during amplification reactions are provided. In particular, methods are provided for converting single primer PCR amplicons to dual primer PCR amplicons.

Single primer to dual primer amplicon switching

Processes, oligonucleotides, and kits for amplifying RNA. In particular, the processes generate and amplify cDNA libraries in which the orientation of the input RNA molecule is preserved in the products. Among the various aspects of the present disclosure is the provision of process for directionally amplifying RNA. The process comprises reverse transcribing at least one RNA molecule in the presence of a plurality of first synthesis primers to generate a plurality of first strands of complementary DNA (cDNA), wherein each of the first synthesis primers comprises a 3' sequence having complementarity to a portion of the RNA molecule, a non-complementary 5&#x2032; sequence corresponding to one or more amplification primers, and optionally an internal tag sequence comprising a first tag sequence.

Directional amplification of RNA

Processes and kits for preparing a plurality of amplification products with reduced non-specific amplification artifacts.

Nucleic acid amplification and library preparation

A composition suitable for formulation of an enzymatic reaction mixture, the composition comprising a reaction component essential for an ex-vivo non-polymerase enzymatic reaction in which a substrate is catalyzed by an enzyme in a reaction mixture to form a product, and a tracer compatible with the enzyme, the composition being substantially free of the substrate.

Tracer reagents that enhance reaction-product analysis

The present invention provides compositions, methods, and kits for protecting thermostable DNA polymerase during amplification reactions conducted at a temperature ranging from about 40&#xb0; C. to greater than 100&#xb0; C. The composition comprises a thermostable DNA polymerase and an anionic detergent or zwitterionic detergent.

Methods of using stabilized compositions of thermostable DNA polymerase and anionic detergent

Stabilized compositions of thermostable DNA polymerase and anionic or zwitterionic detergent

Amplification mixtures, kits, amplicons, kits and processes are provided for amplifying a nucleic acid. In particular, provided are processes which utilize an amplification mixture comprising a polymerase, a deoxynucleotidetriphosphate (dNTP) mixture which contains modified dNTPs, a first primer and a second primer. Further provided are dNTP mixtures which contain modified dNTPs for at least two of the four nucleotide triphosphates, which when incorporated into a polynucelotide, impart resistance to enzymatic degradation by an exonuclease at the sites of incorporation of the modified dNTPs. Also provided are the amplicons and vectors which incorporate the modified nucleotides.

Processes for directionally ligating double stranded nucleic acids

Recombinant DNA processes using a dNTP mixture containing modified nucleotides

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St. Louis, MO, United States of America

Brian W Ward