Methods for incorporating non-perfectly matched oligonucleotides into target-specific hybridization sequences
First Claim
1. A method of making a hybridization probe the method comprising:
- receiving a target-specific hybridization sequence identified by;
a) selecting a candidate target nucleotide sequence in a subject nucleotide sequence;
b) identifying one or more interfering nucleotide sequences, wherein the interfering nucleotide sequences have at least a predetermined number of sequential nucleotides in common with the candidate target nucleotide sequence;
c) identifying a first complementary nucleotide sequence to the target nucleotide sequence;
d) determining a set of replacement positions Pn in the first complementary nucleotide sequence by locating each position at which a G is located;
e) creating a set of second complementary nucleotide sequences by replacing each G at the replacement position with a nucleotide selected from the group consisting of T/U, C, and A, wherein each member of the set of second complementary nucleotide sequences has a single G replaced;
f) characterizing the amount of “
target-second complementary nucleotide sequence”
hybrid formed when the target and second complementary nucleotide sequences are combined in the presence of potentially interfering sequences;
g) identifying the second complementary nucleotide sequence as a symbolic representation of a target-specific hybridization sequence if the amount of “
target-second complementary nucleotide sequence”
hybrid is greater than a predetermined value;
h) determining a set of replacement positions Pn in the first complementary nucleotide sequence by locating each position at which a C or A or T/U if the first complementary nucleotide sequence did not have a G or the amount of “
target-second complementary nucleotide sequence”
hybrid was not greater than the predetermined value;
i) creating the set of second complementary nucleotide sequences by replacing C or A or T/U identified in step h) at the replacement position with a non-complementary nucleotide;
j) characterizing the amount of “
target-second complementary nucleotide sequence”
hybrid formed when the target and second complementary nucleotide sequences are combined in the presence of potentially interfering sequences; and
k) identifying the second complementary nucleotide sequence as the symbolic representation of a target specific hybridization sequence if the amount of “
target-second complementary nucleotide sequence”
hybrid is greater than a predetermined value, and generating an actual nucleotide sequence that is incorporated in the hybridization probe.
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Abstract
The present invention provides a method of designing target-specific hybridization sequences which include one or more mutations. The method of the invention comprises selecting a candidate target nucleotide sequence in a subject nucleotide sequence. A first complementary nucleotide sequence to the target nucleotide sequence is identified by applying the known bonding relationships of the nucleotides. Next, a second complementary nucleotide sequence having one or more mutations is constructed. The amount of the “target-second complementary nucleotide sequence” hybrid formed when the candidate target nucleotide sequence and second complementary nucleotide sequences are combined in the presence of interfering sequences is characterized and used to assess utility of the second complementary nucleotide sequence. The present invention also provides the target-specific hybridization sequences designed by the methods of the invention.
5 Citations
19 Claims
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1. A method of making a hybridization probe the method comprising:
- receiving a target-specific hybridization sequence identified by;
a) selecting a candidate target nucleotide sequence in a subject nucleotide sequence; b) identifying one or more interfering nucleotide sequences, wherein the interfering nucleotide sequences have at least a predetermined number of sequential nucleotides in common with the candidate target nucleotide sequence; c) identifying a first complementary nucleotide sequence to the target nucleotide sequence; d) determining a set of replacement positions Pn in the first complementary nucleotide sequence by locating each position at which a G is located; e) creating a set of second complementary nucleotide sequences by replacing each G at the replacement position with a nucleotide selected from the group consisting of T/U, C, and A, wherein each member of the set of second complementary nucleotide sequences has a single G replaced; f) characterizing the amount of “
target-second complementary nucleotide sequence”
hybrid formed when the target and second complementary nucleotide sequences are combined in the presence of potentially interfering sequences;g) identifying the second complementary nucleotide sequence as a symbolic representation of a target-specific hybridization sequence if the amount of “
target-second complementary nucleotide sequence”
hybrid is greater than a predetermined value;h) determining a set of replacement positions Pn in the first complementary nucleotide sequence by locating each position at which a C or A or T/U if the first complementary nucleotide sequence did not have a G or the amount of “
target-second complementary nucleotide sequence”
hybrid was not greater than the predetermined value;i) creating the set of second complementary nucleotide sequences by replacing C or A or T/U identified in step h) at the replacement position with a non-complementary nucleotide; j) characterizing the amount of “
target-second complementary nucleotide sequence”
hybrid formed when the target and second complementary nucleotide sequences are combined in the presence of potentially interfering sequences; andk) identifying the second complementary nucleotide sequence as the symbolic representation of a target specific hybridization sequence if the amount of “
target-second complementary nucleotide sequence”
hybrid is greater than a predetermined value, and generating an actual nucleotide sequence that is incorporated in the hybridization probe. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
- receiving a target-specific hybridization sequence identified by;
Specification