Methods and kits for highly multiplex single primer extension
The present disclosure provides methods and kits for highly multiplex single primer extensions using a MutS protein and Mg 2+ at a concentration higher than that in a typical PCR reaction. Also disclosed is the use of such methods and kits in next generation sequencing.
1 . A method for performing a multiplex single primer extension reaction, comprising:
(a) extending a plurality of different primers in a single primer extension reaction using a plurality of different double-stranded or partially double-stranded target nucleic acids as templates in a reaction mixture that comprises (1) a MutS protein and (2) Mg 2+ at a minimum concentration of about 6 mM to generate extension products, wherein
the plurality of different primers differs from each other,
the plurality of different primers anneals to only one strand of the different double-stranded or partially double-stranded target nucleic acids, and
the reaction mixture does not comprise a primer that anneals to the other strand of the different double-stranded or partially double-stranded target nucleic acids to exponentially amplify one or more of the different double-stranded or partially double-stranded target nucleic acids or a portion thereof except where most or all of the double-stranded or partially double-stranded target nucleic acids comprise a common sequence at least 10 nucleotides in length, a boosting primer comprising the common sequence or a portion thereof that is at least 10 nucleotides in length that anneals to a sequence complementary to the common sequence or a portion thereof in the other strand of the different double-stranded or partially double-stranded target nucleic acids is optionally present.
2 . The method of claim 1 , wherein the single primer extension reaction is performed in the absence of the boosting primer.
3 . The method of claim 1 , wherein most or all of the target nucleic acids comprise the common sequence at least 10 nucleotides in length, and wherein the reaction is performed in the presence of the boosting primer that comprises the common sequence or a portion thereof that is at least 10 nucleotides in length and anneals to the sequence complementary to the common sequence or a portion thereof in the other strand of the different double-stranded or partially double-stranded target nucleic acids.
4 . The method of claim 1 , wherein each of the different primers is present in the single primer extension reaction at a concentration of no more than 20 nM.
5 . The method of claim 3 , wherein each of the primers other than the boosting primer is present in the single primer extension reaction at a concentration of 1 nM to 10 nM.
6 . The method of claim 1 , wherein Mg 2+ is present in the single primer extension reaction at a concentration of 6 mM to 20 mM.
7 . The method of claim 1 , wherein the plurality of target nucleic acids are genomic DNA or amplification products of genomic DNA.
8 . The method of claim 1 , wherein the plurality of target nucleic acids are cDNA or amplification products of cDNA.
9 . The method of claim 1 , wherein the plurality of target nucleic acids are bisulfite treated genomic DNA or its amplified products.
10 . The method of claim 1 , wherein the MutS protein is a Thermus aquaticus MutS protein.
11 . The method of claim 1 , wherein the primer annealing and extension specificity of the single primer extension reaction is at least 80%.
12 . The method of claim 1 , wherein the average primer annealing and extension efficiency of the single primer extension reaction is at least 10%.
13 . The method of claim 1 , further comprising:
(b) amplifying the extension product(s) to generate amplified extension product(s), and
(c) sequencing the extension product(s) or the amplified extension products.
14 . The method of claim 1 , wherein the target nucleic acids are partially double-stranded, and comprise a double-stranded target nucleic acid sequence and a single-stranded common sequence at the 5′ terminus of each strand of the target nucleic acid sequence, wherein the single-stranded common sequence is at least 10 nucleotides in length.
15 . The method of claim 14 , wherein the target nucleic acids are DNA molecules, the method comprising: prior to step (a),
(i) fragmenting the DNA molecules to generate double-stranded DNA fragments, and
(ii) ligating the single-stranded common sequence to the 5′ terminus of each strand of the double-stranded DNA fragment to provide partially double-stranded target nucleic acids.
16 . The method of claim 1 , wherein each of the different primers comprises at its 5′ terminus a universal sequence that is incapable of specifically annealing to a region of a target nucleic acid.
17 . The method of claim 1 , wherein step (a) is performed in the presence of a high fidelity DNA polymerase.
18 . The method of claim 17 , wherein the high fidelity DNA polymerase is KOD polymerase or Pfu polymerase.
19 . The method of claim 3 , wherein step (a) comprises performing a PCR reaction.