The Perkin-elmer Corporation

The Perkin-elmar Corporation

Nicole M Ellis

Cheryl R Heiner

Katherine D Lazaruk

Patric Sean Walsh

Robert G Kuimelis

Douglas H Smith

Deborah E Dodge

The invention relates to novel chimeric phosphoramidate oligonucleotides and their use in primer-extension methods such as DNA sequencing and nucleic acid amplification. The subject chimeric phosphoramidate oligonucleotides have both N3'-phosphoramidate linkages and phosphodiester linkages. The invention includes methods of primer extension using the subject chimeric oligonucleotides as primers. Primer extension methods of interest include nucleic acid amplification reactions, e.g. PCR, and polynucleotide sequencing reactions. In the primer extension methods of the invention, a chimeric phosphoramidate oligonucleotide primer is annealed to a polynucleotide template. After annealing, the chimeric oligonucleotide primer is extended by joining a nucleotide to the 3' end of the primer by a DNA polymerase catalyzed reaction. Other embodiments of the invention include methods of primer extension using phosphoramidate linkage containing polynucleotide templates. Such methods include annealing an oligonucleotide primer (chimeric or otherwise) to a polynucleotide template comprising at least one phosphoramidate linkage. Nucleotides are added to the primer in a DNA polymerase catalyzed reaction. Primer extension takes place across one or more of the phosphoramidate linkages in the template. The invention includes compositions comprising a chimeric phosphoramidate oligo-nucleotide of the invention and a divalent cation. Divalent cations serve to increase the binding affinity between the chimeric oligonucleotides of the invention and a polynucleotide template. Additionally, divalent cations may be used to stabilize the phosphoramidate linkages of the subject chimeric oligonucleotides against hydrolysis at elevated temperatures, such as the temperatures used in PCR and cycle sequencing. The subject compositions may further comprise a thermostable DNA polymerase such as Taq DNA polymerase.

Phosphoramidate-phosphodiester oligonucleotide chimera as primers

Phosphoramidate-phosphodiester oligonucleotide chimera utilized as

The present invention relates to improved methods of generating polynucleotide sequencing reaction products from cycle sequencing and relates to various instruments and reagents for use in the subject methods. The use of 'step-down' thermal profiles in conjunction with cycle sequencing is described. Step-down thermal profiles are formed by combining several thermal cycle sets such that the annealing temperature of each thermal cycle set is less than the annealing temperature of the preceding thermal cycle set. One embodiment of the invention is a method of generating a plurality of polynucleotide sequencing reaction products in parallel by subjecting a plurality of sequencing solution preparations to a step-down thermal profile, i.e., exposure to repeated thermal cycle sets, each thermal cycle set having an annealing temperature that is lower than the annealing temperature of the annealing phases of the preceding thermal cycle set. Other embodiments of the invention include systems for generating a plurality of polynucleotide sequencing reaction products in parallel. The systems comprise (i) a programmable thermal cycler programmed to perform a step-down thermal profile and (ii) a plurality of polynucleotide sequencing preparations. Additional embodiments of the invention include sets of polynucleotide sequencing reaction preparations that may be used in the methods of the invention. Another aspect of the invention is a device for generating a plurality of polynucleotide sequencing reaction products. The devices of the invention include (i) a plurality of polynucleotide sequencing reaction preparation chambers, each chamber containing a sequencing primer, (ii) a common fluid entry port, and (iii) a fluid dispensing channel, the channel connecting each of the sequencing reaction preparation chambers to the common fluid entry port. At least two of the sequencing primers in the device have Tms different from one another by at least 2.degree. C.

Cycle sequencing thermal profiles

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San Mateo, CA, United States of America

Nicole M Ellis