Preparation of nucleic acid templates for solid phase amplification
First Claim
1. A method of preparing nucleic acid template constructs for solid-phase nucleic acid amplification which comprises performing the following ligation reactions a) and b):
- a) a ligation reaction in which the first end of one or more target polynucleotide molecules of unknown sequence are ligated to surface-bound adaptor polynucleotide molecules; and
b) a ligation reaction in which solution-phase adaptor polynucleotide molecules are ligated to the second end of said target polynucleotide molecules of unknown sequence;
thereby producing one or more template constructs attached to a solid support, wherein each of said template constructs comprises a target polynucleotide molecule of unknown sequence ligated to a surface-bound adaptor polynucleotide molecule and a solution-phase polynucleotide molecule, and wherein the target polynucleotide molecules are not digested with a cleavage reagent prior to step a).
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Abstract
The invention relates to a method of preparing template constructs for solid-phase nucleic acid amplification and to use of the templates in methods of solid-phase nucleic acid amplification. The method involves carrying out two ligation reactions: (a) a ligation reaction in which the first end of one or more target polynucleotide molecules are ligated to surface-bound adaptor polynucleotide molecules, and (b) a ligation reaction in which solution-phase adaptor polynucleotide molecules are ligated to the second end of said target polynucleotide molecules, in order to produce one or more template constructs attached to a solid support.
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Citations
32 Claims
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1. A method of preparing nucleic acid template constructs for solid-phase nucleic acid amplification which comprises performing the following ligation reactions a) and b):
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a) a ligation reaction in which the first end of one or more target polynucleotide molecules of unknown sequence are ligated to surface-bound adaptor polynucleotide molecules; and b) a ligation reaction in which solution-phase adaptor polynucleotide molecules are ligated to the second end of said target polynucleotide molecules of unknown sequence; thereby producing one or more template constructs attached to a solid support, wherein each of said template constructs comprises a target polynucleotide molecule of unknown sequence ligated to a surface-bound adaptor polynucleotide molecule and a solution-phase polynucleotide molecule, and wherein the target polynucleotide molecules are not digested with a cleavage reagent prior to step a). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification