Annealing control primer system for regulating primer annealing specificity and its applications

A process for selectively amplifying a target nucleic acid sequence from a nucleic acid molecule or mixture of nucleic acids, said process comprising carrying out a two-stage polymerase chain reaction (PCR) comprising:

• (1) amplifying the target nucleic acid sequence in a first-stage PCR comprising at least one cycle of primer annealing, primer extending and denaturing, by annealing a pair of annealing control primers to the target nucleic acid sequence at a first annealing temperature, wherein the annealing control primers comprise a 3′-end portion and a 5′-end portion separated by at least one deoxyinosine residue, universal base or non-discriminatory base analog, and wherein the 3′-end portion anneals to the target nucleic acid at the first annealing temperature and wherein the 5′-end portion comprises a universal primer sequence;
• extending the primers to obtain first amplification product;
• denaturing the first amplification product to obtain denatured amplification product;
• and
• (2) re-amplifying the denatured amplification product in a second-stage PCR comprising at least one cycle of annealing, primer extending and denaturing under high stringency conditions, by annealing the universal primer sequence of the 5′-end portion of the annealing control primers to the universal primer sequences located at the 5′ ends of the denatured amplification product and extending the primers to generate second amplification product.

A method for detecting DNA complementary to differentially expressed mRNA in two or more nucleic acid samples comprising:

• (a) providing a first sample of nucleic acids representing a first population of mRNA transcripts and a second sample of nucleic acids representing a second population of mRNA transcripts;
• (b) separately contacting each of said first nucleic acid sample and said second nucleic acid sample with a first annealing control primer, wherein said first annealing control primer has a hybridizing sequence substantially complementary to the differentially expressed mRNA to hybridize therewith;
• (c) reverse transcribing said differentially expressed mRNA to which said first annealing control primer hybridizes to produce a first population of DNA strands that are complementary to said differentially expressed mRNA in said first nucleic acid sample to which said first annealing control primer hybridizes, and a second population of DNA strands that are complementary to said mRNA in said second nucleic acid sample to which said first annealing control primer hybridizes;
• (d) purifying and quantifying each of said first and second populations of complementary DNA strands;
• (e) contacting each of said first and second populations of DNA strands with a second annealing control primer, wherein said second annealing control primer has a hybridizing sequence substantially complementary to said first and second populations of DNA strands;
• (f) extending said second annealing control primer using DNA polymerase to produce a second DNA strand complementary to each of said first and second populations of DNA strands;
• (g) amplifying each second DNA strand obtained from step (f) by at least one PCR cycle comprising denaturing, annealing and primer extension to obtain first and second populations of first amplification products;
• (h) amplifying the first amplification products generated from step (g) by at least one PCR cycle comprising denaturing, annealing and primer extension to generate first and second populations of second amplification products using two universal primers each comprising a sequence corresponding to each 5′-end portion of said first and second annealing control primers;
• and
• (i) comparing the amount of individual amplification products present in said first and second populations of amplification products.