Generation of single-strand circular DNA from linear self-annealing segments

Abstract

The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

Description

[0001] This application is a divisional of U.S. application Ser. No. 09 / 723,685, filed 28 Nov. 2000, which claims the priority of U.S. Provisional Application Ser. No. 60 / 168,511, filed 2 Dec. 1999, the disclosures of which are hereby incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention relates to a process for forming single-stranded circular DNA from single-stranded hairpin segments containing complementary sequences directing spontaneous circularization of said segments with subsequent ligation to form single circles. BACKGROUND OF THE INVENTION [0003] Over the past several decades, research into molecular biology has been greatly enhanced by the plethora of new methodologies made available to workers in the field. Such methods have essentially helped to create the newer area of biotechnology and facilitated its emergence onto the commercial playing field. Especially advantageous has been the methods developed for replicating and amplify...

AI Technical Summary

Benefits of technology

The present invention provides methods for synthesizing single-stranded circular DNA molecules of varying sizes and sequences using pseudocircular DNA segments as a starting material. These methods allow for the formation of enlarged single-stranded circular DNAs containing predetermined nucleotide sequences formed from the sequences of the starting segments. Additionally, the invention provides a means of producing multiple copies of single-stranded DNA circles of varying sizes and of any desired sequences for subsequent mixing to yield a single reaction mixture for further rolling circle replication. The invention also provides hairpin oligonucleotides with complementary ends that can be used to produce single stranded circles without the need for chemical synthesis. Finally, the invention provides hairpin oligonucleotides with coherent ends for ligation to DNA restriction fragments of any desired size and sequence to form circles for amplification by RCA.

Problems solved by technology

Despite the enormous advantages conferred by such technology, there are certain drawbacks.

In addition, the various steps of heating and cooling and the different enzymes required for the process make it expensive, time consuming and cumbersome.

However, PCR remains inadequate for the rapid and inexpensive production of single-stranded circular DNAs.

One of the problems of current RCA technology is that most starting circles are synthesized chemically (to facilitate predetermination of the nucleotide sequence or sequences contained within the circles).

Such synthesis has made production of circles larger than about 100 bases both costly and time consuming.

Of course, circles larger than about 200 nucleotides cannot be effectively prepared using current technology.

Conversely, plasmid technology has not been of much use in this area because the needed starting circles must be single-stranded whereas plasmids are normally duplex DNAs.

The yield of circles by such processes is commonly less than 50% and other, undesired, forms are always present.

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