The present disclosure provides methods, compositions, and kits for performing PCR (including multiplex PCR). The methods, compositions and kits provided herein use one or more primer pairs that contain one or more cleavable bases located at a minimal distance away from the 3′ termini of the primers
The present disclosure provides methods, compositions, and kits for performing PCR (including multiplex PCR). The methods, compositions and kits provided herein use one or more primer pairs that contain one or more cleavable bases located at a minimal distance away from the 3′ termini of the primers, and increase the accuracy of downstream analysis of sequence data.
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1. A method for performing nucleic acid sequencing, comprising: a) amplifying one or more different target nucleic acids in the presence of one or more different primer pairs specific to the one or more different target nucleic acids in a single reaction mixture via polymerase chain reaction (PCR),w
1. A method for performing nucleic acid sequencing, comprising: a) amplifying one or more different target nucleic acids in the presence of one or more different primer pairs specific to the one or more different target nucleic acids in a single reaction mixture via polymerase chain reaction (PCR),wherein each primer of the one or more different primer pairs contains one or more cleavable bases, and wherein in substantially all of the primers of the one or more different primer pairs, none of the one or more cleavable bases is less than 4 nucleotides away from the 3′ terminus of the primer that comprises the one or more cleavable bases,b) cleaving the one or more cleavable bases in the amplification product(s) of step a) to produce single-stranded DNA overhangs in the amplification product(s),c) digesting the single stranded DNA overhangs obtained in step b) to generate trimmed amplification product(s),d) ligating adapters to the trimmed amplification product(s) to produce adapter-linked trimmed amplification product(s), ande) sequencing the adapter-linked trimmed amplification product(s) of step d). 2. The method of claim 1, wherein step a) comprises amplifying a single target nucleic acid in the presence of a primer pair specific to the single target nucleic acid in the single reaction mixture. 3. The method of claim 1, wherein step a) comprises amplifying a plurality of different primer pairs specific to the plurality of different target nucleic acids in the single reaction mixture. 4. The method of claim 1, wherein in all of the primers of the one or more different primer pairs, each of one or more cleavable bases is at least 4, 5, 6, 7, or 8 nucleotides away from the 3′ terminus of the primer that comprises the one or more cleavable bases. 5. The method of claim 1, wherein the cleavable base is uracil. 6. The method of claim 1, wherein the cleavable base is inosine, an oxidized pyrimidine, an oxidized purine, 5-hydroxyuracil, 5-hydroxylmethyluracil, or 5-formyluracil. 7. The method of claim 1, wherein the one or more different primer pairs comprise at least 100 different primer pairs. 8. The method of claim 1, wherein in substantially all of the primers of the different primer pairs, none of the one or more cleavable bases is less than 5 nucleotides away from the 3′ terminus of the primer that comprises the one or more cleavable bases. 9. The method of claim 1, wherein in substantially all of the primers of the different primer pairs, none of the one or more cleavable bases is less than 6 nucleotides away from the 3′ terminus of the primer that comprises the one or more cleavable bases.
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