Nucleic acid constructs and methods of use
The present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like.
1. A method for identifying a sequence variant in an analyte nucleic acid fragment, comprising:
(a) attaching a unique identifier oligonucleotide from a pool of unique identifier oligonucleotides to an end of each analyte nucleic acid fragment of a plurality of analyte nucleic acid fragments using primer extension to form a plurality of uniquely identified analyte nucleic acid fragments, wherein the number of different unique identifiers in the pool of unique identifier oligonucleotides is greater than the number of different analyte nucleic acid fragments during primer extension;
(b) redundantly determining a nucleotide sequence of a uniquely identified analyte nucleic acid fragment, wherein the determined nucleotide sequences which share a unique identifier oligonucleotide form a family of members; and
(c) identifying a nucleotide sequence as accurately representing an analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least two families,
wherein a sequence variant in the analyte nucleic acid fragment is identified when the nucleotide sequence that accurately represents the analyte nucleic acid fragment is different from a reference sequence.
2. The method of claim 1 , wherein the nucleotide sequence is identified as accurately representing an analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least five families.
3. The method of claim 1 , wherein the nucleotide sequence is identified as accurately representing an analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least ten families.
4. The method of claim 1 , wherein the sequence variant comprises an allelic variant.
5. The method of claim 1 , wherein the sequence variant comprises a polymorphism.
6. The method of claim 1 , wherein the sequence variant comprises a mutation.
7. The method of claim 1 , wherein the sequence variant comprises a nucleotide substitution.
8. The method of claim 1 , wherein the sequence variant comprises a nucleotide insertion.
9. The method of claim 1 , wherein the sequence variant comprises a nucleotide deletion.
10. The method of claim 1 , further comprising determining nucleotide sequences of a plurality of members of a family.
11. The method of claim 10 , further comprising comparing nucleotide sequences of the family.
12. The method of claim 1 , wherein the identified nucleotide sequence is free of a synthesis or sequencing error.
13. The method of claim 1 , wherein the attaching step comprises performing a polymerase chain reaction (PCR).
14. The method of claim 1 , further comprising isolating analyte nucleic acid fragments comprising the same unique identifier oligonucleotide.
15. The method of claim 1 , wherein the unique identifier oligonucleotides are about 10, about 20, about 30 or more bases.
16. The method of claim 1 , wherein each uniquely identified analyte nucleic acid fragment comprises one or more unique identifier oligonucleotide and one or more universal priming site for subsequent amplification.
17. A method for identifying a sequence variant in an analyte nucleic acid fragment, comprising:
(a) attaching a unique identifier oligonucleotide from a pool of unique identifier oligonucleotides to an end of each analyte nucleic acid fragment of a plurality of analyte nucleic acid fragments using a primer in an amplification reaction to form a plurality of uniquely identified analyte nucleic acid fragments, wherein the number of different unique identifiers in the pool of unique identifier oligonucleotides is greater than the number of different analyte nucleic acid fragments during amplification;
(b) amplifying the uniquely identified analyte nucleic acid fragments to form a family of nucleotide sequences from each uniquely identified analyte nucleic acid fragment; and
(c) identifying a nucleotide sequence as accurately representing an analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least two families,
wherein a sequence variant in the analyte nucleic acid fragment is identified when the nucleotide sequence that accurately represents the analyte nucleic acid fragment is different from a reference sequence.
18. A method for identifying a substitution, an insertion, or a deletion in an analyte nucleic acid fragment, comprising:
(a) attaching a unique identifier oligonucleotide from a pool of unique identifier oligonucleotides to an end of each analyte nucleic acid fragment of a plurality of analyte nucleic acid fragments using amplification primers to form a plurality of uniquely identified analyte nucleic acid fragments, wherein the number of different unique identifiers in the pool of unique identifier oligonucleotides is greater than the number of different analyte nucleic acid fragments during amplification, whereby each uniquely identified analyte nucleic acid fragment comprises one or more unique identifier oligonucleotide and one or more universal priming site for subsequent amplification;
(b) amplifying the uniquely identified analyte nucleic acid fragments to form a family of uniquely identified analyte nucleic acid fragments from each uniquely identified analyte nucleic acid fragment;
(c) determining nucleotide sequences of a plurality of members of a family of uniquely identified analyte nucleic acid fragments;
(d) comparing nucleotide sequences of the family of uniquely identified analyte nucleic acid fragments;
(e) identifying a nucleotide sequence as accurately representing an analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least two families; and
(f) identifying a substitution, an insertion, or a deletion in the analyte nucleic acid fragment when the nucleotide sequence that accurately represents the analyte nucleic acid fragment is different from a reference sequence by a substitution, an insertion, or a deletion in the analyte nucleic acid fragment.
19. The method of claim 18 , wherein the amplification primers are PCR primers.
20. The method of claim 18 , wherein each uniquely identified analyte nucleic acid fragment comprises two or more unique identifier oligonucleotides.
21. The method of claim 18 , wherein the amplification primers comprise a phosphorothioate linkage.
22. The method of claim 18 , wherein the nucleotide sequence is identified as accurately representing the analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least five families.
23. The method of claim 18 , wherein the nucleotide sequence is identified as accurately representing the analyte nucleic acid fragment when the nucleotide sequence is a consensus sequence of a plurality of members of a family and the nucleotide sequence is found in at least ten families.