Methods and compositons for bi-directional polymorphism detection

A method for identifying one or more nucleotides present at a polymorphic site on one or more alleles comprising the steps of:

• a) obtaining an upper strand of target nucleic acids from the one or more alleles and a lower strand of target nucleic acids from the one or more alleles;
• wherein each strand comprises the polymorpic site;
• b) hybridizing an upper strand primer that is complementary to the upper strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the upper strand of target nucleic acids so as to obtain one or more unpaired nucleotide bases to be identified on the upper strand, and hybridizing a lower strand primer that is complementary to the lower strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the lower strand so as to obtain one or more unpaired nucleotide bases to be identified on the lower strand;
• the upper and lower strand primers each have a unique tag at the 5′ end capable of binding to known positions on a solid support;
• c) exposing the hybridized upper and lower strand primers to a polymerization agent in a mixture comprising one or more nucleotides so that one or more primer extension products are formed if the one or more nucleotides in the mixture is complementary to the polymorphic site on the upper strand or lower strand of target nucleic acids;
• d) contacting the solid support with the mixture so as to cause each unique sequence tag to bind to known positions on the solid support;
• and
• e) detecting each bound primer, wherein the positions of the primers on the solid support in conjunction with any one or more primer extension products allows identification of the polymorphic site on the one or more alleles.

A method for identifying one or more nucleotides present at a polymorphic site on the one or more alleles comprising the steps of:

• a) obtaining an upper strand of target nucleic acids from the one or more alleles and a lower strand of target nucleic acids from the one or more alleles, wherein each strand comprises the polymorphic site;
• b) hybridizing an upper strand primer that is complementary to the upper strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the upper strand of target nucleic acids so as to obtain an unpaired nucleotide base to be identified at the polymorphic site on the upper strand, and hybridizing a lower strand primer that is complementary to the lower strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the lower strand so as to obtain an unpaired nucleotide base to be identified at the polymorphic site on the lower strand;
• wherein the upper and lower primers each have a unique tag at the 5′ end capable of binding to known positions on a solid support;
• and
• c) exposing the hybridized upper and lower strand primers to a polymerization agent in a mixture comprising at least four different terminating nucleotides so as to form primer extension products wherein the primers are extended bidirectionally when the terminating nucleotide in the mixture is complementary to the polymorphic site on the upper strand or lower strand of target nucleic acids;
• wherein at least two different terminating nucleotides have the same detectable characteristic;
• d) contacting the solid support with the mixture so as to cause each unique sequence tag to bind to known positions on the solid support;
• and
• e) detecting each bound primer, wherein the positions of the primers on the solid support in conjunction with any detectable characteristic allows identification of the polymorphic site on the one or more alleles.

A method for identifying one or more nucleotides present at a polymorphic site on one or more alleles comprising the steps of:

• a) obtaining an upper strand of target nucleic acids from the one or more alleles and a lower strand of target nucleic acids from the one or more alleles, wherein each strand comprises the polymorphic site;
• b) hybridizing an upper strand primer that is complementary to the upper strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the upper strand of target nucleic acids so as to obtain one or more unpaired nucleotide bases to be identified on the upper strand, and hybridizing a lower strand primer that is complementary to the lower strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the lower strand so as to obtain one or more unpaired nucleotide bases to be identified on the lower strand;
• c) exposing the hybridized upper and lower strand primers to a polymerization agent in a mixture comprising one or more nucleotides so that one or more primer extension products are formed if the one or more nucleotides in the mixture are complementary to the polymorphic site on the upper strand or lower strand of target nucleic acids wherein the primers are extended bidirectionally;
• and
• d) separating any one or more primer extension products from unextended primers so as to identify the polymorphic site on the one or more alleles.

A method for identifying one or more nucleotides present at a polymorphic site on one or more alleles comprising the steps of:

• a) obtaining an upper strand of target nucleic acids from the one or more alleles and a lower strand of target nucleic acids from the one or more alleles, wherein each strand comprises a polymorphic site;
• b) hybridizing an upper strand primer that is complementary to the upper strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the upper strand of target nucleic acids so as to obtain one or more unpaired nucleotide bases to be identified on the upper strand, and hybridizing a lower strand primer that is complementary to the lower strand of target nucleic acids at a region immediately adjacent to the polymorphic site on the lower strand so as to obtain one or more unpaired nucleotide bases to be identified on the lower strand;
• c) exposing the hybridized upper and lower strand primers to a polymerization agent in an extension mixture comprising one or more nucleotides so that one or more primer extension products are formed if the one or more nucleotides in the extension mixture is complementary to the polymorphic site on the upper strand or lower strand of target nucleic acids;
• and
• d) detecting in the extension mixture one or more nucleotides not incorporated into the one or more primer extension products so as to identify the one or more nucleotides present at the polymorphic site.