Patent ID: 7547514
Filing Date: 2009-06-16
Classification: C12Q

Abstract:
1. A method of exponentially amplifying DNA, the method comprising the steps of: (a) forming a polymerase chain reaction amplification mixture comprising the DNA, multiple copies of a single gene profiling (SGP) primer consisting of a primer sequence consisting of 6-9 nucleotides and other amplification reagents at appropriate concentrations, wherein the SGP primer binds to multiple sites on the DNA and functions as a forward primer and a reverse primer; (b) amplifying the DNA by a first polymerase chain reaction of the amplification mixture, wherein the first polymerase chain reaction comprises 10-100 cycles of denaturing, annealing and elongation steps to produce a first amplification product in the amplification mixture, wherein the elongation steps are performed for a first length of time, wherein the first amplification product comprises (i) first linear amplification products, wherein each of the first linear amplification products has a 5′ end and a 3′ end, contains the primer sequence at the 5′ end, does not contain a reverse complement of the primer sequence at the 3′ end and is single-stranded, and (ii) first exponential amplification products, wherein each of the first exponential amplification products has a 5′ end and a 3′ end, contains the primer sequence at the 5′ end, contains a reverse complment of the primer sequence at the 3′ end, and is double stranded, and wherein the first expontial products are produced during each cycle after the first cycyle of the first polmerase chain reaction; and (c) amplifying the first exponential amplification products by a second polymerase chain reaction of the amplification mixture, wherein the second polymerase chain reaction comprises one or more cycles of denaturing, annealing and elongation steps to produce a second amplification product in the amplification mixture, wherein the elongation steps are performed for a second length of time that is about 40%-60% of the first length of time, wherein the second amplification product comprises (i) second linear amplification products derived from a first portion of the first exponential amplification products, wherein each of the second linear amplification products has a 5′ end and a 3′ end, contains the primer sequence at the 5′ end, does not contain a reverse complement of the primer sequence at the 3′ end, is shorter in length than the first exponential amplification product from which it is derived and is single-stranded and (ii) second exponential amplification products derived from a second portion of the first exponential amplification products, wherein each of the second exponential amplification products has a 5′ end and a 3′ end, contains the primer sequence at the 5′ end, contains a reverse complement of the primer sequence at the 3′ end, is the same length as the first exponential amplification product from which it is derived and is double stranded and wherein the second exponential products are produced during each cycle after the first cycle of the second polymerase chain reaction, wherein the reverse complement of the primer sequence is fully complementary to the primer sequence.