Abstract: Methods employing internal oligonucleotide standards in isothermal nucleic acid amplification reactions to determine the efficacy of the amplification reaction and to quantify pre-amplification target levels.

Abstract: Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.

Abstract: The invention is a method for generating nucleic acid sequences ends which comprises;(a) hybridizing a primer to a nucleic acid sequence,(b) hybridizing a primer to the nucleic acid sequence in (a) located 5' to the primer in (a), and(c) polymerizing both primers so that the primer in (a) is displaced from the nucleic acid sequence.The invention provides a method for generating target nucleic acid sequences for subsequent amplification. The method is applicable to both DNA and RNA.