Patent ID: 6114155
Filing Date: 2000-09-05
Classification: C12Q

Abstract:
A method for performing Gap-LCR (Gapfilling Chain Reaction) which comprises:a) forming a reaction medium comprising a target nucleic acid sequence, a DNA polymerase, a first nucleic acid sequence probe, a second nucleic acid sequence probe, a ligating enzyme selected from ligase or an enzyme with a ligase function, and at least one additional nucleoside-containing reactant, wherein said target nucleic acid sequence comprises a gap sequence which is flanked on one side by a region complementary to said first nucleic acid sequence probe and on the other side by a region complementary to said second nucleic acid probe;b) adding to the reaction medium an internal control sequence comprising a gap sequence flanked on one side by a region complementary to said first nucleic acid sequence probe and on the other side by a region complementary to said second nuclcic acid probe, wherein the gap sequence in the internal control sequence differs from the gap sequence in the target nucleic acid sequence such that the internal control sequence either contains a restriction endonuclease site not found in the target nucleic acid sequence, or lacks a restriction endonuclease site found in the target nucleic acid sequence;c) amplifying the target nucleic acid sequence and the internal control sequence using the first nucleic acid sequence probe and the second nucleic acid sequence probe, wherein during amplification the first probe and the second probe hybridize to said regions complementary to said first and second probes in both the target nucleic acid sequence and the internal control sequence;andd) detecting amplification products produced in step c), wherein internal control sequence amplification products either contain a restriction endonuclease site not found in target nucleic acid sequence amplification products, or lack a restriction endonuclease site found in target nucleic acid sequence amplification products; ande) optionally, comparing internal control sequence amplification products with target nucleic acid sequence amplification products by digesting amplification products with a restriction endonuclease.