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 detecting a first genetic element and a second genetic element on a common double-stranded nucleic acid molecule, the method comprising:
performing a first amplification reaction in a first amplification reaction mixture, wherein the first amplification reaction mixture comprises: i) the common double-stranded nucleic acid molecule, wherein the common double-stranded nucleic acid molecule comprises a first strand and a second strand, and also the first genetic element and the second genetic element, wherein the first genetic element comprises a first genetic element first complementary sequence and a first genetic element second complementary sequence, wherein the second genetic element comprises a second genetic element first complementary sequence and a second genetic element second complementary sequence, wherein the first genetic element first complementary sequence and the second genetic element first complementary sequence are part of the first strand, and wherein the first genetic element second complementary sequence and the second genetic element second complementary sequence are part of the second strand; ii) a first amplification reaction first primer, wherein the first amplification reaction first primer has a nucleotide sequence which is complementary to the first genetic element first complementary sequence; and iii) a first amplification reaction second primer, wherein the first amplification reaction second primer has a nucleotide sequence which is complementary to the second genetic element second complementary sequence, and wherein a first amplification reaction product is generated in the first amplification reaction mixture, wherein the first amplification reaction product comprises at least a portion of the first genetic element and at least a portion of the second genetic element, and wherein the first amplification reaction product has a double-stranded, linear configuration;
performing a ligation reaction in a ligation reaction mixture, wherein the ligation reaction mixture comprises i) the first amplification reaction product; and ii) a ligase enzyme, and wherein in the ligation reaction mixture a circular ligation product is formed from the first amplification reaction product, wherein the circular ligation product is double-stranded and comprises a circular ligation product first strand and a circular ligation product second strand, and wherein the circular ligation product first strand comprises the nucleotides of the first amplification reaction product first strand and the circular ligation product second strand comprises the nucleotides of the first amplification reaction product second strand; and
performing a second amplification reaction in a second amplification reaction mixture, wherein the second amplification reaction mixture comprises: i) the circular ligation product; ii) a second amplification reaction first primer, wherein the second amplification reaction first primer has a nucleotide sequence which is complementary to the first genetic element second complementary sequence; and iii) a second amplification reaction second primer, wherein the second amplification reaction second primer has a nucleotide sequence which is complementary to the second genetic element first complementary sequence, and wherein a second amplification reaction product is generated in the second amplification reaction mixture, wherein the second amplification reaction product comprises at least a portion of the first genetic element and at least a portion of the second genetic element; and
detecting the second amplification reaction product.
2 . The method of claim 1 , wherein the first genetic element and the second genetic element are separated from each other on the double-stranded nucleic acid molecule by at least 2000 and no more than 50000 nucleotides.
3 . The method of claim 1 , wherein the first genetic element and the second genetic element are separated from each other on the double-stranded nucleic acid molecule by at least 4000 and no more than 50000 nucleotides.
4 . The method of claim 1 , wherein the first amplification reaction is a polymerase-chain reaction (PCR) amplification reaction.
5 . The method of claim 1 , wherein the first amplification reaction first primer and the first amplification reaction second primer are each at least 6 nucleotides and no more than 50 nucleotides in length.
6 . The method of claim 1 , wherein at least one of the first amplification reaction first primer and the first amplification reaction second primer in the first amplification reaction mixture is phosphorylated at the 5′ end of the primer.
7 . The method of claim 1 , wherein both of the first amplification reaction first primer and the first amplification reaction second primer in the first amplification reaction mixture are phosphorylated at the 5′ end of the primer.
8 . The method of claim 1 , further comprising, prior to performing the ligation reaction, incubating the first amplification reaction product with a kinase enzyme.
9 . The method of claim 8 , wherein the kinase enzyme is T4 polynucleotide kinase.
10 . The method of claim 1 , wherein the ligase enzyme is T4 DNA ligase.
11 . The method of claim 1 , wherein the second amplification reaction first primer and the second amplification reaction second primer are each at least 6 nucleotides and no more than 60 nucleotides in length.
12 . The method of claim 1 , wherein the second amplification reaction is PCR.
13 . The method of claim 1 , wherein the second amplification reaction is performed without thermocycling.
14 . The method of claim 13 , wherein the second amplification reaction first primer comprises a first region and a second region, wherein the second region of the second amplification reaction first primer is complementary to the second genetic element first complementary sequence and the second amplification reaction second primer comprises a first region and a second region, wherein the second region of the second amplification reaction second primer is complementary to the first genetic element second complementary sequence, and wherein the first region of the second amplification reaction second primer is complementary to the first region of the second amplification reaction first primer.
15 . The method of claim 14 , wherein the first region of the second amplification reaction second primer is also complementary to at least a portion of the first strand of the circular ligation product.
16 . The method of claim 1 , wherein the second amplification reaction product is detected in real-time as it is generated.
17 . The method of claim 1 , wherein the first genetic element is an antibiotic resistance gene.
18 . The method of claim 1 , wherein the second genetic element is a pathogen gene.
19 . The method of claim 1 , wherein the first genetic element is the mecA gene.
20 . The method of claim 1 , wherein the second genetic element is a gene from Staphylococcus aureus.
21 - 40 . (canceled)