Methods of nucleic acid amplification and sequencing
First Claim
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1. A method of amplifying one or more nucleic acid templates on a solid support which comprises:
- a) bringing into contact the following components under conditions which permit a nucleic acid amplification reaction;
i) a solid support,ii) a plurality of forward and reverse amplification primers, wherein the solid support is provided with the forward and/or reverse amplification primers immobilised thereon, andiii) one or more nucleic acid templates to be amplified comprising at the 3′
end a primer-binding sequence which is a sequence of nucleotides capable of annealing to the forward amplification primers and at the 5′
end a primer-binding sequence which is a sequence of nucleotides the complement of which is capable of annealing to the reverse amplification primers; and
b) carrying out a nucleic acid amplification reaction whereby said template(s) is/are amplified with said forward and reverse amplification primers,characterised in that the amplification primers immobilised on the solid support comprise a template-specific portion which is a sequence of at least 26 consecutive nucleotides capable of annealing to a primer binding sequence in the template or the complement thereof and that the forward and reverse primers are not capable of annealing to any part of the template other than their respective primer binding sequences during the nucleic acid amplification reaction.
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Abstract
The invention relates to a method of amplifying one or more nucleic acid templates on a solid support in a nucleic acid amplification reaction, for example by solid-phase PCR using one or more amplification primers attached to the solid support. The method is characterised in that the amplification primers used comprise a template-specific portion which is a sequence of at least 26 consecutive nucleotides and are not capable of annealing to target regions in the template under conditions of the amplification reaction. The method is particularly useful for amplifying human genomic DNA.
28 Citations
44 Claims
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1. A method of amplifying one or more nucleic acid templates on a solid support which comprises:
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a) bringing into contact the following components under conditions which permit a nucleic acid amplification reaction; i) a solid support, ii) a plurality of forward and reverse amplification primers, wherein the solid support is provided with the forward and/or reverse amplification primers immobilised thereon, and iii) one or more nucleic acid templates to be amplified comprising at the 3′
end a primer-binding sequence which is a sequence of nucleotides capable of annealing to the forward amplification primers and at the 5′
end a primer-binding sequence which is a sequence of nucleotides the complement of which is capable of annealing to the reverse amplification primers; andb) carrying out a nucleic acid amplification reaction whereby said template(s) is/are amplified with said forward and reverse amplification primers, characterised in that the amplification primers immobilised on the solid support comprise a template-specific portion which is a sequence of at least 26 consecutive nucleotides capable of annealing to a primer binding sequence in the template or the complement thereof and that the forward and reverse primers are not capable of annealing to any part of the template other than their respective primer binding sequences during the nucleic acid amplification reaction. - 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, 29)
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28. (canceled)
- 30. A solid support having immobilised thereon a plurality of forward and/or reverse amplification primers, characterised in that said forward and/or reverse amplification primers comprise a template-specific portion capable of annealing to the template or the complement thereof which is a sequence of at least 26 consecutive nucleotides.
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44. (canceled)
Specification