Diagnostic methods and compositions
Methods and compositions for the identification of genetic-related information are provided. At least portions of methods provided herein may be performed without thermocycling. Methods and compositions may include reagents such as nucleic acid polymerases and primers.
1. A method for assessing the identity of a nucleotide at a position of interest in a nucleotide sequence in a polynucleotide template, the method comprising:
A) generating multiple copies of a polynucleotide template in a polymerase chain reaction (PCR) amplification reaction mixture, wherein the PCR amplification reaction mixture comprises a PCR amplification reaction first primer and a PCR amplification reaction second primer, wherein in the PCR amplification reaction mixture, the PCR amplification reaction first primer anneals to the polynucleotide template and the PCR second primer anneals to a polynucleotide which is complementary to the polynucleotide template, and wherein in the PCR amplification reaction mixture, multiple copies of a PCR amplification reaction product are formed, wherein the PCR amplification reaction product is a double-stranded nucleic acid molecule comprising a first strand and a second strand, and wherein a first strand of the PCR amplification reaction product is a copy of the polynucleotide template;
B) providing copies of the PCR amplification reaction product generated in step A) in each of at least a non-thermocycling first reaction mixture and a non-thermocycling second reaction mixture, wherein:
the polynucleotide template comprises a first portion, a second portion and a third portion, wherein the third portion is situated in the polynucleotide template between the first portion and the second portion and wherein the position of interest is in the third portion;
the non-thermocycling first reaction mixture comprises copies of the polynucleotide template, a non-thermocycling first primer, and a non-thermocycling second primer, wherein:
the non-thermocycling first primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template;
the non-thermocycling second primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template;
the first region of the non-thermocycling first primer is complementary to the first region of the non-thermocycling second primer; and
the first region of the non-thermocycling second primer is complementary to the third portion of the polynucleotide template;
the second reaction mixture comprises copies of the polynucleotide template, a non-thermocycling third primer, and a non-thermocycling fourth primer, wherein:
the non-thermocycling third primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to the first portion of the polynucleotide template;
the non-thermocycling fourth primer comprises a first region and a second region, wherein the first region comprises a 5′ end of the primer, the second region comprises a 3′ end of the primer, and the second region is complementary to a sequence which is complementary to a second portion of the polynucleotide template;
the first region of the non-thermocycling third primer is complementary to the first region of the non-thermocycling fourth primer; and
the first region of the non-thermocycling fourth primer is complementary to the third portion of the polynucleotide template; and
the nucleotide sequence of the first region of the non-thermocycling second primer differs from the nucleotide sequence of first region of the non-thermocycling fourth primer by a single nucleotide, wherein the position of the different nucleotide in the non-thermocycling second and non-thermocycling fourth primers corresponds to the position of the nucleotide of interest in the polynucleotide template when the nucleotide sequence of the first region of the non-thermocycling second primer or non-thermocycling fourth primer is oriented with the nucleotide sequence of the third portion of the polynucleotide template for maximum complementation of the sequences;
C) incubating the non-thermocycling first reaction mixture and non-thermocycling second reaction mixture under conditions without thermocycling; and
D) comparing the rate or amount of amplification of the polynucleotide template in the non-thermocycling first reaction mixture to the rate or amount of amplification of the polynucleotide template in the non-thermocycling second reaction mixture, wherein the rate or amount of amplification of the polynucleotide template is indicative of the degree of complementation between first region of the non-thermocycling second or non-thermocycling fourth primer and the nucleotide sequence of the third portion of the polynucleotide template.
2. The method of claim 1 , wherein the PCR amplification reaction first primer is at least 10 and no more than 80 nucleotides in length, and wherein when the PCR amplification reaction first primer is annealed to the polynucleotide template, at least 3 nucleotides of the PCR amplification reaction first primer are mis-matched according to Watson-Crick base-pairing rules with corresponding nucleotides on the polynucleotide template.
3. The method 1 , wherein the PCR amplification reaction first primer is at least 10 and no more than 80 nucleotides in length, and wherein when the PCR amplification reaction first primer is annealed to the polynucleotide template, at least 5 nucleotides of the PCR amplification reaction first primer are mis-matched according to Watson-Crick base-pairing rules with corresponding nucleotides on the polynucleotide template.
4. The method of claim 1 , wherein the PCR amplification reaction second primer is at least 10 and no more than 80 nucleotides in length, and wherein when the PCR amplification reaction second primer is annealed to the polynucleotide which is complementary to the polynucleotide template, at least 3 nucleotides of the PCR amplification reaction first primer are mis-matched according to Watson-Crick base-pairing rules with corresponding nucleotides on the polynucleotide which is complementary to the polynucleotide template.
5. The method claim 1 , wherein the PCR amplification reaction second primer is at least 10 and no more than 80 nucleotides in length, and wherein when the PCR amplification reaction second primer is annealed to the polynucleotide which is complementary to the polynucleotide template, at least 5 nucleotides of the PCR amplification reaction first primer are mis-matched according to Watson-Crick base-pairing rules with corresponding nucleotides on the polynucleotide which is complementary to the polynucleotide template.
6. The method claim 1 , wherein the position of interest in the nucleotide sequence in the polynucleotide template is a SNP.
7. The method of claim 1 , wherein the polynucleotide template is from the hepatitis C virus.
8. The method of claim 7 , wherein the polynucleotide template is from the hepatitis C NS3 gene.
9. The method of claim 8 , wherein the position of interest in the nucleotide sequence in the polynucleotide template is in the codon encoding 80 th amino acid of the NS3 gene.
10. The method of claim 1 , wherein a concatemer strand comprising at least three copies of the polynucleotide template is generated during the incubation of the non-thermocycling reaction mixture.