Improved fidelity of thermostable ligases for detection of microsatellite repeat sequences using nucleoside analogs.
Microsatellite repeats consisting of dinucleotide sequences are ubiquitous in the human genome and have proven useful for linkage analysis, positional cloning and forensic identification purposes. In this study, the potential of utilizing the ligase detection reaction for the analysis of such microsatellite repeat sequences was investigated. Initially, the fidelity of thermostable DNA ligases was measured for model dinucleotide repeat sequences. Subsequently, the effect of modified oligonucleotides on ligation fidelity for dinucleotide repeats was determined using the nucleoside analogs nitroimidazole, inosine, 7-deazaguanosine and 2-pyrimidinone, as well as natural base mismatches. The measured error rates for a standard dinucleotide template indicated that the nitroimidazole nucleoside analogs could be used to increase the fidelity of ligation when compared to unmodified primers. Furthermore, use of formamide in the ligation buffer also increased ligation fidelity for dinucleotide repeat sequences. Using ligation-based assays to detect polymorphic alleles of microsatellite repeats in the human genome opens the possibility of using array-based typing of these loci for human identification, loss-of-heterozygosity studies and linkage analysis.
Zirvi
M
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Department of Microbiology, Box 62, Hearst Microbiology Research Center, Strang Cancer Prevention Center, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Bergstrom
D E
DE
Saurage
A S
AS
Hammer
R P
RP
Barany
F
F
D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers; D018900:Dinucleotide Repeats; D004795:Enzyme Stability; D015894:Genome, Human; D006151:Guanosine / Q000031:analogs & derivatives; D006801:Humans; D007288:Inosine; D009593:Nitroimidazoles; D009705:Nucleosides / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011743:Pyrimidines
D017931:DNA Primers; D009593:Nitroimidazoles; D009705:Nucleosides; D011743:Pyrimidines; D006151:Guanosine; D007288:Inosine; C068218:7-deazaguanosine; D011088:DNA Ligases