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Method of nucleic acid amplification

  • US 8,476,044 B2
  • Filed: 04/16/2008
  • Issued: 07/02/2013
  • Est. Priority Date: 04/01/1997
  • Status: Expired due to Fees
First Claim
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1. A method of simultaneously sequencing a population of random nucleic acid molecules of unknown sequence, comprising:

  • (a) attaching known ends to said population of random nucleic acids thereby forming a plurality of nucleic acid targets each of said nucleic acid targets comprising a first part having a sequence capable of annealing to a first amplification primer, a second part having a sequence complementary to a sequence capable of annealing to a second amplification primer, and a third part comprising a random unknown sequence located between the first and second parts, wherein the known ends are attached to each of the random nucleic acids in the population;

    (b) dispersing the plurality of nucleic acid targets such that individual target molecules of the plurality of nucleic acid targets are separated from each other; and

    (c) performing an amplification reaction using first and second amplification primers, wherein the first amplification primer anneals to the first part of each of said nucleic acid targets, the second amplification primer anneals to a sequence complementary to the second part of each of said nucleic acid targets and wherein at least said first or said second amplification primers are immobilized at different locations on a single solid support, such that each of the plurality of nucleic acid targets is simultaneously amplified thereby producing 1000 or more different nucleic acid colonies of immobilized amplified nucleic acid molecules, a plurality of said immobilized amplified nucleic molecules in each of said colonies having the same sequence, and wherein the amplification reaction results in amplification of each of the random nucleic acids in the population; and

    (d) simultaneously sequencing the immobilized amplified nucleic acid molecules present in said 1000 or more different nucleic acid colonies such that said population of random nucleic acid molecules of unknown sequence are simultaneously sequenced.

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