End modification to prevent over-representation of fragments
First Claim
1. A method of reducing representation of the ends of a primary polynucleotide sequence in a library of fragments generated from said primary polynucleotide sequence, the method comprising:
- a) performing an amplification reaction with a modified forward primer and a modified reverse primer to introduce a modification at or near each 5′
terminus of a double-stranded primary polynucleotide sequence, said modification configured to prevent fragments originating from the ends of the primary polynucleotide from ligating to an adapter, thereby forming a modified primary polynucleotide sequence;
b) fragmenting the modified primary polynucleotide sequence such that the 5′
terminal fragment and the 3′
terminal fragment each comprise the modification;
c) ligating an adapter to fragments generated in step b), thereby forming a library of fragments generated from said primary polynucleotide sequence.
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Accused Products
Abstract
The invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides and also the use of the 5′ and 3′ modified library of templates in methods of solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, wherein over-representation of “end” sequences of the primary polynucleotide molecules from when the 5′ and 3′ modified library is generated is greatly reduced or prevented.
11 Citations
17 Claims
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1. A method of reducing representation of the ends of a primary polynucleotide sequence in a library of fragments generated from said primary polynucleotide sequence, the method comprising:
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a) performing an amplification reaction with a modified forward primer and a modified reverse primer to introduce a modification at or near each 5′
terminus of a double-stranded primary polynucleotide sequence, said modification configured to prevent fragments originating from the ends of the primary polynucleotide from ligating to an adapter, thereby forming a modified primary polynucleotide sequence;b) fragmenting the modified primary polynucleotide sequence such that the 5′
terminal fragment and the 3′
terminal fragment each comprise the modification;c) ligating an adapter to fragments generated in step b), thereby forming a library of fragments generated from said primary polynucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of reducing representation of the ends of a primary polynucleotide sequence in a library of fragments generated from said primary polynucleotide sequence, the method comprising:
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a) introducing a modification at or near each 5′
terminus of a double-stranded primary polynucleotide sequence, wherein said modification is a modified deoxynucleoside triphosphate introduced using a nucleotide polymerase, said modification configured to prevent fragments originating from the ends of the primary polynucleotide from ligating to an adapter, thereby forming a modified primary polynucleotide sequence;b) fragmenting the modified primary polynucleotide sequence such that the 5′
terminal fragment and the 3′
terminal fragment each comprise the modification;c) ligating an adapter to fragments generated in step b), thereby forming a library of fragments generated from said primary polynucleotide sequence. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
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Specification