Constant cluster seeding

Abstract

The invention provides methods for controlling the density of different molecular species on the surface of a solid support. A first mixture of different molecular species is attached to a solid support under conditions to attach each species at a desired density, thereby producing a derivatized support having attached capture molecules. The derivatized support is treated with a second mixture of different molecular species, wherein different molecular species in the second mixture bind specifically to the different capture molecules attached to the solid support. One or more of the capture molecules can be reversibly modified such that the capture molecules have a different activity before and after the second mixture of molecular species are attached. In particular embodiments, the different molecular species are nucleic acids that are reversibly modified to have different activity in an amplification reaction.

Description

[0001]This application is based on, and claims the benefit of, U.S. Provisional Application Ser. No. 61 / 035,254, filed Mar. 10, 2008 and entitled “Constant Cluster Seeding,” the entirety of which is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The current invention relates to the field of nucleic acid amplification. More specifically, the present invention provides methods for optimising the density of nucleic acid clusters produced on a solid support whilst eliminating the need for multiple sample titration steps.BACKGROUND TO THE INVENTION[0003]Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.[0004]A number of methods for high throughput nucleic acid sequencing rely on a universal amplification reaction, whereby a DNA sample is randomly fragmented, then treat...

AI Technical Summary

Benefits of technology

[0012]In a particular aspect, the invention provides a method of controlling the density of colonies of amplified single stranded polynucleotides formed on a solid support. The method can include the steps of (a) providing a plurality of single stranded template polynucleotides; (b) providing a plurality of at least three oligonucleotides immobilised to a solid support wherein at least one of the oligonucleotides is a capture sequence capable of hybridising to the single stranded template polynucleotides, and at least two of the oligonucleotides are amplification sequences which are incapable of hybridising to the single stranded template polynucleotides, wherein the capture sequences are immobilised at a lower density than the amplification sequences; (c) applying the single stranded template polynucleotides to the solid support under suitable condition

Problems solved by technology

The generation of a plurality of primer pairs for each individual template can be cumbersome and expensive for complex mixtures of templates.

If the density of clusters is too great, it may be difficult to individually resolve each cluster and overlapping colonies may be formed.

However, such titrations can lead to a loss of valuable flow cell channels due to a cluster density that is too high or too low, a loss of template sample, an increase in the level of reagents required or increase in sample processing time.

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