Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1998-01-15
2000-09-05
Myers, Carla J.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 912, 435 915, 435 9151, C12P 1934, C12Q 168
Patent
active
061141550
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns an internal control for use in the "gap-filing ligase chain reaction" (Gap-LCR). The internal control being a synthetic nucleotide, has substantially the same length and contains the hybridization sites for the same four LCR-probes as the target nucleic acid (the target sequence). The internal control and the target become accordingly amplified in a GAP-LCR with substantially the same effectiveness. Additionally the synthetic oligonucleotide is modified in the gap between the LCR-probes in such a way that it comprises a unique site for a restriction enzyme. Treatment of the LCR product with such a restriction enzyme will cut the internal control LCR product so that the LCR product from the target may be analyzed separately. The disclosed internal control may be used both in qualitative and quantitative LCR-based analysis.
BACKGROUND
Analyses based on enzymatic amplification of nucleic acids sometimes give false negative results e.g. on account of the presence of inhibitors in the samples. An inhibitor may for instance keep the enzymes from performing the reactions effectively. Samples containing inhibitors may be erroneously considered to lack a given target since their analyses does not give any signal. An addition of an internal control to the samples will reveal the presence of inhibitors since it is expected that the internal control always will be amplified and will give a signal. The internal control functions thus as a qualitative control.
The use of an internal control in polymerase chain reaction (PCR) based analyses has been disclosed by Matsumara et al. (Jpn. J. Clin. Oncol. 1992, 22:335-341). Their PCR with two sets of primers amplify a target of interest, but simultaneously another target functioning as an internal control in the reaction is amplified. Becker and Hahlbroeck (Nucl. Acid Res. 1989, 17:9437-9446) disclose a PCR based on a procedure wherein both target and internal control DNA is amplified by using the same two primers. Their internal control has become mutated in vitro so that it contains a unique site for a restriction enzyme. The PCR product from the internal control may be cleaved with the restriction enzyme and then be separated from the PCR product from the target by gel electrophoresis. Gilliland et al. (Proc. Natl. Acad Sci. USA 1990, 87:2725-2729) and Ursi et al. (APMIS 1992, 100:635-639) discloses internal controls which contain extra blocks of DNA between the PCR primer-sites. After PCR-amplification by using the same two primers the PCR-product from the internal control will have a different length than the target PCR product. These two may either be separated by gel electrophoresis or by hybridization with two selective probes. Also for "nucleic acid sequence amplification" (the NASBA system) there has been disclosed by Kievits and Lens (WO 94/04706) a procedure wherein both target and a longer/shorter internal control become amplified simultaneously by using the same primers.
Both the PCR and the NASBA internal controls may be used for quantitative analyses, for instance by densiometric measurements of the two bands appearing in gel electrophoresis of the amplification product.
So far ligase chain reaction (LCR) based analyses have lacked an internal control to be used in quantitative analyses. In a standard LCR disclosed by Backman (European Patent Office - 320.380 - 1987) the amplification of the target is being performed with only DNA ligase. In the reaction four probes are hybridised in pairs adjacent to each other on each strand in the target DNA. The nicks between the probes are closed by the DNA ligase so that two new DNA strands are produced. These may be used as a target in the next cycle of the process, see FIG. 1a.
The LCR internal control disclosed by Griffiths and Emery (WO 93/02215) has a different sequence than the target DNA on one side of the nick. The LCR amplification of the internal control requires thus the use of two extra LCR probes. These hybridise to the unique part of the control DNA, whereas the two LCR pro
REFERENCES:
Birkenmeyer et al., "Preliminary evaluation of the ligase chain reaction for specific detection of Neisseria gonorrhoeae", J. Clin. Micro., 30:3089-94 (1992).
Abravaya, K. et al. Nucleic Acids Research 23(4):675-682, Feb. 1995.
Berg Einar Sverre
Skaug Kjell
Johannsen Diana
Myers Carla J.
Nath Gary M.
Statens Institutt for Folkehelse
Yellin Deborah H.
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