ENRICHMENT OF NUCLEIC ACID TARGETS
Methods and apparatus providing improved fidelity and specificity when separating nucleic acids from a sample, but without need for amplification. In particular, using the disclosed methods, it is possible to isolate a variant nucleic acid (i.e., a mutation) from a non-target nucleic acid (i.e., a wild-type) when the variant is present in the original sample at a much lower concentration than the non-target, e.g., 1:10,000 , without substantial loss of the variant.
1 . A method for enriching a sample, the method comprising the steps of:
obtaining a sample comprising:
a non-target nucleic acid comprising a non-target sequence; and
a plurality of nucleic acids comprising a target sequence;
wherein the target sequence and the non-target sequence differ by an insertion, a deletion, or a polymorphism at a position of interest;
wherein the plurality of nucleic acids comprising the target sequence are between 200 bases and 1000 bases in total length; and
wherein X is a ratio of the plurality of nucleic acids to the non-target nucleic acid in the sample;
enriching for the plurality of nucleic acids comprising the target sequence by applying a time-varying driving field to the sample in the presence of a separation medium;
wherein Y is a ratio of the plurality of nucleic acids to the non-target nucleic acid in the sample, after enrichment, and Y is at least 10 times greater than X.
2 . The method of claim 1 , wherein the non-target nucleic acid the plurality of nucleic acids comprising the target sequence have different methylation or acetylation patterns.
3 . The method of claim 1 , wherein the sample is a biological sample.
4 . The method of claim 3 , wherein the biological sample comprises tissue, blood, sputum, sweat, urine, feces, tears, aspirate, or a combination thereof.
5 . The method of claim 3 , wherein the biological sample is from a human.
6 . The method of claim 1 , further comprising identifying the target sequence.
7 . The method of claim 6 , wherein identifying comprises:
sequencing at least a portion of one or more of the plurality of nucleic acids comprising the target sequence;
contacting one or more of the plurality of nucleic acids comprising the target sequence with a labeled moiety; or
an amplification-based detection.
8 . The method of claim 1 , wherein presence of the target sequence is indicative of a disorder.
9 . The method of claim 8 , wherein the disorder is a genetic disorder.
10 . The method of claim 8 , wherein the disorder is cancer.
11 . The method of claim 1 , wherein Y is at least 100 times greater than X.
12 . The e hod of claim 11 , wherein Y is at least 1,000 times greater than X.
13 . The method of claim 1 , wherein enriching comprises applying a periodic electric field to the sample.
14 . The method of claim 13 , further comprising periodically changing the temperature of the medium.
15 . The method of claim 1 , wherein the non-target sequence is a wild-type sequence.
16 . The method of claim 1 , wherein at least 20% of the plurality of nucleic acids comprising the target sequence in the sample is recovered after enrichment.
17 . The method of claim 16 , wherein at least 50% of the plurality of nucleic acids comprising the target sequence in the sample is recovered after enrichment.
18 . The method of claim 1 , wherein the sample is simultaneously enriched for a plurality of nucleic acids comprising more than one target sequence.