A Ligase-Mediated Gene Detection Technique

Abstract

An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule. The two oligonucleotides are then joined covalently by the action of a DNA ligase, provided that the nucleotides at the junction are correctly base-paired. Thus single nucleotide substitutions can be distinguished. This strategy permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.

Detection of single base changes in nucleic acids

“…However, as not all mutations alter restriction enzyme sites the coverage of the screen is obviously incomplete. It has been suggested to be less than 50 % (Landegren et al, 1988a). Despite this, harmless mutations (polymorphisms or RFLPs) have been detected after screening using a battery of restriction enzymes and used in tracking disease carrying genes in families (Wolfe, 1988;Kan & Dozy, 1978 Restriction enzymes have been effective in studying viral variants (Griffiths & Grundy, 1987;Tenover, 1988) as enough variation occurs between isolates to be sure that differences will be found with a small number of enzymes.…”

“…There are several such systems, some of which are based on the principles and methods discussed above in the screening methods. This problem of the need for simpiicity has been addressed by Nicholls & Malcolm (1989) and Landegren et al (1988a).…”

“…However, little evidence was provided of differences in the rate of ligation between DNA containing and not containing a mismatch at the ligation site. Recently, conditions have been found that allow only minimal ligation when a mismatch occurs either side of the point of ligation, paving the way for a simple method of assaying for the presence of a particular mutation (Landegren et al, 1988a;Alves & Carr, 1988;Wu & Wallace, 1989b). The system used has been a continuous strand of DNA to which two adjacent oligonucleotides are hybridized with 3' and 5' bases of each abutting, so that ifjoined the region would be complementary to the continuous strand (Fig.…”

“…1 d). Under conditions of high salt and spermidine various bases (Landegren et al, 1988a) A similar system was developed by Wu & Wallace (1 989a) with high salt and spermidine. They extended the above work by testing the effect of mismatches on either side of the ligation point and showed that discrimination could be achieved in either case from matched pairs on either side.…”

Using high-throughput SNP technologies to study cancer

“…The GoldenGate Assay is a combination of an oligonucleotide ligation (OLA) (Landegren et al, 1988) and allele-specific extension reaction analysed with the Illumina core technology, which analyses thousands of differentially labeled beads simultaneously. For each SNP, three oligonucleotides are synthesized; two oligos are allele specific (ASO) with a universal PCR primer sequence located at the 5 0 end (P1 and P2).…”