Systems and methods for determining genetic data
Systems and methods of polynucleotide sequencing are provided. Systems and methods optimize control, speed, movement, and/or translocation of a sample (e.g., a polynucleotide) within, through, or at least partially through a nanopore or a type of protein or mutant protein in order to accumulate sufficient time and current blocking information to identify contiguous nucleotides or plurality of nucleotides in a single-stranded area of a polynucleotide.
1. A system for sequencing a single stranded nucleic acid molecule, the system comprising:
(a) a nucleic acid molecule comprising a plurality of single stranded regions and a plurality of double stranded regions, wherein:
(a1) each of the plurality of double stranded regions has a melting temperature of at least 30° C., and is formed by hybridizing a portion of the plurality of single stranded regions to an oligonucleotide analog from a plurality of oligonucleotide analogs comprising at least about 25% universal bases; and
(a2) each of the plurality of single stranded regions comprises a portion that does not hydbridize to the oligonucleotide analog; and
(b) a nanopore that permits passage of each of the plurality of single stranded regions of the nucleic acid molecule and does not permit passage of each of the plurality of double stranded regions of the nucleic acid molecule;
(c) a sensing electrode adjacent to the nanopore, such that the sensing electrode is capable of monitoring a current that passes through the nanopore, wherein the universal bases are capable of pairing with each of A, C, T and G in a sequence-independent manner.
2. The system of claim 1 , wherein the at least about 25% universal bases comprises at least about 50% of the universal bases.
3. The system of claim 1 , wherein the at least about 25% universal bases comprises at least about 80% of the universal bases.
4. The system of claim 1 , wherein the oligonucleotide analogs further comprise a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a morpholino, or any combination thereof.
5. The system of claim 1 , wherein the universal base comprises 5-nitroindole, 3-nitropyrrole, 3-methyl 7-propynyl isocarbostyril (PIM), 3-methyl isocarbostyril (MICS), 5-methyl isocarbostyril (SMICS), or any combination thereof.
6. The system of claim 1 , wherein the at least about 25% universal bases comprise at least 3 of the universal bases.
7. The system of claim 1 , wherein the at least about 25% universal bases comprise at least 5 of the universal bases.
8. The system of claim 1 , wherein the at least about 25% universal bases comprise at least 10 of the universal bases.
9. The system of claim 1 , wherein the plurality of oligonucleotide analogs comprises at least one of adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U).
10. The system of claim 1 , wherein the nanopore is in a membrane that is adjacent to the sensing electrode.
11. The system of claim 1 , wherein the sensing electrode is coupled to an integrated circuit.