SAMPLE PREPARATION FOR NUCLEIC ACID AMPLIFICATION
Presented are methods and compositions for preparing samples for amplification and sequencing. Particular embodiments relate to methods of obtaining nucleic acids material directly from tissues such as whole blood or FFPE samples.
1 . A method of performing a solid phase amplification reaction, comprising amplifying a target nucleic acid using at least one of an immobilized forward and an immobilized reverse primer wherein the amplification reaction is performed in the presence of one or more of formalin, paraffin, cellular components, protein, proteinase, extracellular matrix components, collagen, and tissue debris.
2 . The method of claim 1 , wherein the amplification reaction is performed in the presence of at least 0.001 pg paraffin.
3 . The method of claim 1 , wherein the amplification reaction is performed in the presence of at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg paraffin.
4 . The method of claim 1 , wherein the proteinase comprises proteinase K.
5 . The method of claim 4 , wherein the amplification reaction is performed in the presence of at least 0.001 pg proteinase K.
6 . The method of claim 4 , wherein the amplification reaction is performed in the presence of at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg proteinase K.
7 . A flowcell comprising immobilized amplification primers and one or more of formalin, paraffin, cellular components, protein, proteinase, extracellular matrix components, collagen, and tissue debris.
8 . The flowcell of claim 7 , wherein the flowcell comprises at least 0.001 pg paraffin.
9 . The flowcell of claim 7 , wherein the flowcell comprises at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg paraffin.
10 . The flowcell of claim 7 , wherein the proteinase comprises proteinase K.
11 . The flowcell of claim 10 , wherein the flowcell comprises at least 0.001 pg proteinase K.
12 . The flowcell of claim 10 , wherein the flowcell comprises at least 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, or at least 1 mg proteinase K.
13 . A method for preparing a sample for sequencing comprising placing a formalin-fixed paraffin embedded tissue sample into contact with an amplification reaction buffer and performing targeted amplification directly on the FFPE sample, without first extracting nucleic acids from the tissue sample.
14 . The method of claim 13 , wherein there is an incubation period after combining the sample and the lysis reagent and before the amplification step.
15 . The method of claim 13 , wherein the steps of lysing the sample and amplifying the nucleic acid contained therein is conducted in a single pot reaction.
16 . The method of claim 13 , wherein the time taken from combining the sample and lysis reagent to beginning the amplification process is less than about 20 minutes, preferably less than about 15 minutes, preferably less than about 10 minutes, preferably less than about 5 minutes, preferably less than about 4 minutes, preferably less than about 3 minutes, preferably less than about 2 minutes, preferably between about 1-5 minutes, preferably between about 2-4 minutes.