Qiagen Gmbh

Molecular Staging Inc.

Gyanendra Kumar

Rajanikanth Bandaru

Michael Egholm

David C Ward

Michael J Lane

Patricia Bray-Ward

Patricio Abarzúa

Disclosed are compositions and methods for amplification of nucleic acid sequences of interest with greater efficiency and fidelity. The disclosed method relates to isothermal amplification techniques, such as Multiple Displacement Amplification (MDA), where the generation of DNA artifacts is decreased or eliminated. Generally, this can be accomplished by carrying out the reaction at elevated temperature. In particularly useful embodiments of the method, sugars and/or other additives can be used to stabilized the polymerase at high temperature. It has been discovered that generation of high molecular weight artifacts, in an isothermal amplification procedure, is substantially reduced or eliminated while still allowing the desired amplification of input DNA by carrying out the reaction at a higher temperature and, optionally, in the presence of one or more additives. It also has been discovered that isothermal amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed at a higher temperature and, optionally, in the presence of one or more additives.

Method of whole genome amplification with reduced artifact production

Disclosed are compositions and methods for detecting small quantities of analytes such as proteins and peptides. The method involves associating a DNA circle with the analyte and subsequent release and rolling circle replication of the circular DNA molecule. In the method, an amplification target circle is associated with analytes using a conjugate of the circle and a specific binding molecule that is specific for the analyte to be detected. Amplification target circles not associated with the proteins are removed, the amplification target circles that are associated with the proteins are decoupled from the specific binding molecule and amplified by rolling circle amplification. The amplification is isothermic and can result in the production of a large amount of nucleic acid from each primer. The amplified DNA serves as a readily detectable signal for the analytes.

Detection method using dissociated rolling circle amplification

Disclosed are compositions and methods for amplification of RNA molecules. The disclosed method involves synthesizing first strand cDNA molecules from RNA molecules, circularizing the first strand cDNA molecules and replicating the circularized first strand cDNA molecules using rolling circle replication. The method can be aided by the use of specialized primers for cDNA synthesis and specialized probes for circularizing the first strand cDNA molecules. The method can be used to replicate and amplify multiple RNA molecules, such as all RNA molecules in a sample or all mRNA molecules in a sample, or be used to replicate and amplify specific RNA molecules. Rolling circle replication of the circularized first strand cDNA molecules results in long DNA strands containing tandem repeats of the cDNA sequence. The tandem sequence DNA can be used directly (for detection of sequences, for example), further amplified, or used any other purpose. Double-stranded tandem sequence DNA can be used to produce unit lengths of the cDNA sequence. Tandem sequence DNA can also be transcribed to produce transcripts having sequence complementary to or matching the sequence of RNA molecules.

Rolling circle amplification of RNA

The present invention provides a novel method for ligation of oligonucleotides containing 5&prime;-phosphorothioates on complementary templates by the action of DNA ligases. This reaction is readily applied to the synthesis of a single stranded circular DNA containing a phosphorothioate linkage at the site of ligation junction. The efficiency of 5&prime;-phosphorothioate directed ligation reaction by ATP dependent DNA ligase reaction is similar to conventional 5&prime;-phosphate ligation. The utility of enzymatic ligation in probing specific sequences of DNA is also described. The present invention also provides a novel non-enzymatic ligation of 5&prime;-phosphorothioates that has been applied to the synthesis of single strand phosphorothioate and phosphate circular DNA. A process for detecting the presence of a mismatch in an otherwise complementary pair of oligonucleotides is disclosed using an enzyme-based technique which shows the presence of a mismatch by failing to form a ligated single stranded DNA circle that can optionally be amplified using standard methods of rolling circle amplification.

5&prime;-thio phosphate directed ligation of oligonucleotides and use in detection of single nucleotide polymorphisms

Disclosed is a method and compositions for the sensitive detection of the amount and location of specific nucleic acid sequences. The method makes use of a branched oligomer, referred to as a lollipop oligomer, that has a tail portion, a right arm portion, and a left arm portion. These three components are joined at a common junction making a three-tailed structure. The two arms each end with sequences complementary to adjacent sequences in a target sequence. This allows the right and left arms to be ligated together when the oligomer is hybridized to the target sequence, thus topologically linking the oligomer to the target sequence. The tail portion can then be detected at the location of the target sequence.

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Guilford, CT, United States of America

Gyanendra Kumar