Nucleic acid detection and amplification by chemical linkage of oligonucleotides

A process for linear amplification and detection, in a sample containing a nucleic acid of interest, of a target nucleic acid molecule including a target sequence, the process comprising the steps of:

• (a) contacting an oligonucleotide probe pair with stretches of nucleotide bases present in the nucleic acids of interest, wherein:
• (i) said oligonucleotide probe pair includes an oligonucleotide probe 1 and an oligonucleotide probe 2;
• (ii) said oligonucleotide probe 1 includes a targeting sequence A and said oligonucleotide probe 2 includes a targeting sequence C;
• (iii) one of said oligonucleotides includes a protecting sequence B;
• (iv) said targeting sequence A of oligonucleotide probe 1 and said targeting sequence C of oligonucleotide probe 2 are complementary to adjacent portions of the target sequence;
• (v) said targeting sequence A' of oligonucleotide probe 1' and said targeting sequence C' of oligonucleotide probe 2' are complementary to adjacent portions of the target complementary sequence;
• (vi) said protecting sequence B does not hybridize to the target sequence when said targeting sequence A and targeting sequence C hybridize to the target sequence;
• (vii) said protecting sequence B' does not hybridize to the target complementary sequence when said targeting sequence A' and said targeting sequence C' hybridize to the target complementary sequence;
• (viii) a chemical functionality group X1 is attached to a sugar or base moiety of the last nucleotide in said sequence A of oligonucleotide probe 1, a chemical functionality group Y1 is attached to a sugar or base moiety of the last nucleotide in said sequence C of oligonucleotide probe 2, said chemical functionality group X1 is reactive with said chemical functionality group Y1;
• (ix) a chemical functionality group X2 is attached to a sugar or base moiety of the last nucleotide in said sequence A' of oligonucleotide probe 1', a chemical functionality group Y2 is attached to a sugar or base moiety of the last nucleotide in said sequence C' of oligonucleotide probe 2', said chemical functionality group X2 is reactive with said chemical functionality group Y2;
• (x) when said targeting sequence A and targeting sequence C hybridize to the target sequence, said chemical functionality group X1 reacts with said chemical functionality group Y1 to form a chemical bond, and a complementary strand of joined oligonucleotide product is formed, whereas when said targeting sequence A' and targeting sequence C' hybridize to the target complementary sequence, said chemical functionality group X2 reacts with said chemical functionality group Y2 to form an alternative chemical bond, a complementary strand of joined oligonucleotide product is formed;
• (b) providing hybridization conditions permitting targeting sequence A of oligonucleotide probe 1 and targeting sequence C of oligonucleotide probe 2 to hybridize with adjacent portions of the target sequence and permitting targeting sequence A' of oligonucleotide probe 1' and targeting sequence C' of oligonucleotide probe 2' to hybridize with adjacent portions of the target complementary sequence;
• (c) providing conditions permitting joining of said oligonucleotide probe 1 and oligonucleotide probe 2, hybridized after step (b) to said adjacent portions of the target sequence to each other by forming said chemical bond between said chemical functionality groups X1 and Y1, thereby forming said joined oligonucleotide product having the target complementary sequence, and joining of said oligonucleotide probe 1' and oligonucleotide probe 2', hybridized after step (b) to said adjacent portions of the target complementary sequence to each other by forming said alternative chemical bond between said chemical functionality groups X2 and Y2, thereby forming said joined oligonucleotide product having the target sequence;
• (d) treating the sample under a denaturing condition;
• (f) repeating steps (b) through (d) a desired number of times;
• and
• (g) detecting said joined oligonucleotide product.

A process for amplifying and detecting, in a sample containing a nucleic acid of interest, a nucleic acid molecule including a wild type target sequence, a mutant target sequence, a wild-type target complementary sequence and/or a mutant target complementary sequence, in a single or double stranded form, the process comprising the steps of:

• (a) contacting a first, second, third and fourth oligonucleotide probe complement pairs with stretches of nucleotide bases present in the nucleic acids of interest, wherein:
• (i) said first oligonucleotide probe complement pair includes oligonucleotide probe 1 and oligonucleotide probe 1', said second oligonucleotide probe complement pair includes oligonucleotide probe 2 and oligonucleotide probe 2', said third oligonucleotide probe complement pair includes oligonucleotide probe 3 and oligonucleotide probe 3' and said fourth oligonucleotide probe complement pair includes oligonucleotide probe 4 and oligonucleotide probe 4';
• (ii) said oligonucleotide probes 1 and 3 each includes a targeting sequence A, said oligonucleotide probes 1' and 3' each includes a targeting sequence A', said oligonucleotide probes 2 and 4 each includes a targeting sequence C, said oligonucleotide probes 2' and 4' each includes a targeting sequence C';
• (iii) one of said oligonucleotides of each of said first and second oligonucleotide probe pairs includes a protecting sequence B and B', respectively, whereas one of said oligonucleotides of each of said third and fourth oligonucleotide probe pairs includes a protecting sequence D and D', respectively;
• (iv) said targeting sequence A of oligonucleotide probe 1 and said targeting sequence A' of oligonucleotide probe 1' are complementary to each other, said targeting sequence A of oligonucleotide probe 3 and said targeting sequence A' of oligonucleotide probe 3' are complementary to each other;
• (v) said targeting sequence C of oligonucleotide probe 2 and said targeting sequence C' of oligonucleotide probe 2' are complementary to each other, said targeting sequence C of oligonucleotide probe 4 and said targeting sequence C' of oligonucleotide probe 4' are complementary to each other;
• (vi) said oligonucleotide probes 1 and 2 form a first oligonucleotide pair such that said targeting sequence A of oligonucleotide probe 1 and said targeting sequence C of oligonucleotide probe 2 are complementary to adjacent portions of the wild type target sequence, said oligonucleotide probes 3 and 4 form a third oligonucleotide pair such that said targeting sequence A of oligonucleotide probe 3 and said targeting sequence C of oligonucleotide probe 4 are complementary to adjacent portions of the mutant target sequence;
• (vii) said oligonucleotide probes 1' and 2' form a second oligonucleotide pair such that said targeting sequence A' of oligonucleotide probe 1' and said targeting sequence C' of oligonucleotide probe 2' are complementary to adjacent portions of the wild type target complementary sequence, said oligonucleotide probes 3' and 4' form a fourth oligonucleotide pair such that said targeting sequence A' of oligonucleotide probe 3' and said targeting sequence C' of oligonucleotide probe 4' are complementary to adjacent portions of the mutant target complementary sequence;
• (viii) said protecting sequences B, B', D and D' does not hybridize to the nucleic acid of interest when said targeting sequences hybridize to the nucleic acid of interest;
• (x) a chemical functionality group X1 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence A of oligonucleotide probe 1, a chemical functionality group Y1 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence C of oligonucleotide probe 2, said chemical functionality group X1 is reactive with said chemical functionality group Y1, a chemical functionality group X3 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence A of oligonucleotide probe 3, a chemical functionality group Y3 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence C of oligonucleotide probe 4, said chemical functionality group X3 is reactive with said chemical functionality group Y3;
• (xi) a chemical functionality group X2 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence A' of oligonucleotide probe 1', a chemical functionality group Y2 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence C' of oligonucleotide probe 2', said chemical functionality group X2 is reactive with said chemical functionality group Y2, a chemical functionality group X4 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence A' of oligonucleotide probe 3', a chemical functionality group Y4 is attached to a sugar or base moiety of the last nucleotide in said targeting sequence C' of oligonucleotide probe 4', said chemical functionality group X4 is reactive with said chemical functionality group Y4;
• (xii) when said targeting sequence A and said targeting sequence C hybridize to the wild type target sequence, said chemical functionality group X1 reacts with said chemical functionality group Y1 to form a first chemical bond, and a wild type complementary strand of joined oligonucleotide product is formed, when said targeting sequence A' and said targeting sequence C' hybridize to the wild type target complementary sequence, said chemical functionality group X2 reacts with said chemical functionality group Y2 to form a second chemical bond, and a wild type complementary strand of joined oligonucleotide product is formed, when said targeting sequence A and said targeting sequence C hybridize to the mutant target sequence, said chemical functionality group X3 reacts with said chemical functionality group Y3 to form a third chemical bond, and a mutant complementary strand of joined oligonucleotide product is formed, whereas when said targeting sequence A' and said targeting sequence C' hybridize to the mutant target complementary sequence, said chemical functionality group X4 reacts with said chemical functionality group Y4 to form a fourth chemical bond, and a mutant complementary strand of joined oligonucleotide product is formed;
• (b) providing hybridization conditions permitting said targeting sequence A of oligonucleotide probe 1 and said targeting sequence C of oligonucleotide probe 2 to hybridize with said adjacent portions of the wild type target sequence, permitting said targeting sequence A' of oligonucleotide probe 1' and said targeting sequence C' of oligonucleotide probe 2' to hybridize with said adjacent portions of the wild type target complementary sequence, permitting said targeting sequence A of oligonucleotide probe 3 and said targeting sequence C of oligonucleotide probe 4 to hybridize with said adjacent portions of the mutant target sequence and permitting said targeting sequence A' of oligonucleotide probe 3' and said targeting sequence C' of oligonucleotide probe 4' to hybridize with said adjacent portions of the mutant target complementary sequence;
• (c) providing conditions permitting:
• (i) joining of said oligonucleotide probe 1 and oligonucleotide probe 2 to each other by forming said first chemical bond between said chemical functionality groups X1 and Y1, thereby forming said first joined oligonucleotide product having the wild type target complementary sequence;
• and
• (ii) joining of said oligonucleotide probe 3 and oligonucleotide probe 4 to each other by forming said third chemical bond between said chemical functionality groups X3 and Y3, thereby forming said third joined oligonucleotide product having the mutant target complementary sequence;
• (d) providing conditions permitting:
• (i) joining of said oligonucleotide probe 1' and oligonucleotide probe 2' to each other by forming said second chemical bond between said chemical functionality groups X2 and Y2, thereby forming said second joined oligonucleotide product having the wild type target sequence;
• and
• (ii) joining of said oligonucleotide probe 3' and oligonucleotide probe 4' to each other by forming said fourth chemical bond between said chemical functionality groups X4 and Y4, thereby forming said fourth joined oligonucleotide product having the mutant target sequence;
• (e) treating the sample under a denaturing condition;
• (f) repeating steps (b) through (e) a desired number of times;
• and
• (g) detecting the joined oligonucleotide products.