Method, substrate and device for separating nucleic acids

A method comprising:

• applying a sample comprising a target nucleic acid having a first molecular weight to a sample application zone of a substrate, wherein the substrate is a membrane and wherein the target nucleic acid having the first molecular weight is substantially immobilized at the sample application zone;
• laterally flowing a nucleic acid amplification reaction mixture comprising a deoxyribonucleic acid (DNA) polymerase across a length of the substrate through the sample application zone such that a nucleic acid amplification product having a second molecular weight is formed by amplifying the target nucleic acid, wherein one or more PCR inhibitors are present in the sample application zone, and wherein the one or more PCR inhibitors present in the sample application zone are washed away by said laterally flowing the nucleic acid amplification reaction mixture comprising the DNA polymerase across the length of the substrate through the sample application zone prior to said amplifying the target nucleic acid;
• and
• separating the target nucleic acid having the first molecular weight from the nucleic acid amplification product having the second molecular weight by said laterally flowing the nucleic acid amplification reaction mixture comprising the DNA polymerase across the length of the substrate through the sample application zone such that the nucleic acid amplification product having the second molecular weight is migrated away from the sample application zone,
• wherein the target nucleic acid having the first molecular weight is amplified and separated from the nucleic acid amplification product having the second molecular weight without a separate washing step, and wherein the first molecular weight is higher than the second molecular weight.