Internal standards for quantitative competitive PCR

Details Internal standards for quantitative competitive PCR Internal standards for quantitative competitive PCR

1996-10-25

1998-10-20

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

536 221, 536 253, 435 911, C12P 1934, C07H 2104, C07H 2102, C07H 2100

Patent

active

058245162

ABSTRACT:
The present invention relates to a method for constructing internal standards used in competitive polymerase chain reaction (PCR) for determining the amount of a target nucleic acid sequence in a sample. The method comprises the steps of a) digesting the cloned target nucleic acid sequence with a cohesive-end generating restriction endonuclease; b) filling the restriction endonuclease cohesive-ends of step a) by Klenow fragment of DNA polymerase; and c) blunt-end ligating the fragment of nucleic acid of step b) to form the internal standard. The internal standard nucleic acid sequence differs from the target nucleic acid sequence such that a restriction endonuclease recognition site is destroyed by adding sufficient contiguous bases to the target nucleic acid sequence such that the size difference between the target nucleic acid sequence and the internal standard nucleic acid sequence can be detected by gel electrophoresis.

REFERENCES:
patent: 5219727 (1993-06-01), Wang
Sidhu et al. Competitor internal standards for quantitative detection of mycoplasma DNA, vol. 128, pp. 207-212, 1995.
Li, H.H. et al., 1988, Nature, 335:414-417.
Rappolee, D.A., et al., 1988, Science 241:708-712.
Gilliland, G. et al., 1990, Proc. Natl. Acad. Sci. USA, 87:2725-2729.
Forster E., 1994, BioTechnique, 16(1):18-20.

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