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Biacore’s SPR technology as a mutation screening toolReprinted from BIAjournal 8 (1) 2001

Alan McWhirter and 1Kazuhiro Nakatani

Biacore AB, Uppsala, Sweden1Department of SyntheticChemistry and BiologicalChemistry, Faculty ofEngineering, Kyoto University,Kyoto, Japany.

The volume of sequence

information from the human

genome project demands the

development of a tool to rapidly

scan DNA for mutations. Such a

technology would also be valuable

in cancer research as a tool to

detect early mutation events. Today,

single nucleotide polymorphisms

(SNPs), are routinely detected using

methods that rely largely on the

separation of DNA fragments by

gel electrophoresis or HPLC,

neither of which are readily suited

to high throughput analysis.

This review discusses how Biacore’s

SPR technology may come to fill

this role.

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BIACORE is a registered trademark of Biacore AB. Copyright © Biacore AB 2001

Kazuhiro Nakatani and colleagues atKyoto University have used Biacore's SPRtechnology as an alternative means tosingle nucleotide polymorphism (SNP)analysis. They designed and synthesized alow molecular weight ligand thatspecifically binds double stranded DNAcontaining G:G mismatches arising fromnative C:G pairings and which has minimalreactivity with any of the other sevenpossible mismatch combinations (A:A,A:C, A:G, T:T, T:C, T:G or C:C) (1).

The ligand, naphthyridine, is modelled torecognize the characteristic open bulge thatis created between DNA strands at the siteof a G:G mismatch, at which nocomplementary base (cytosine) is presentwith which to form a hydrogen-bondedbase pair. Naphthyridine is synthesized as adimer and is therefore able to formsimultaneous hydrogen bonds with guanineon both DNA strands.

Experiments showed that the meltingtemperature, at which half of the doublestranded DNA in a sample dissociates intosingle chains and which is a measure ofDNA stability, increased by 16.4ºC in thepresence of dimeric, but not monomericnaphthyridine, much higher than for nativeDNA or DNA of an otherwise identicalcomposition containing other mismatches.

Naphthyiridine contains an aminoalkylchain and can therefore be covalentlylinked by amine coupling to carboxylgroups on a Biacore® CM5 chip. 27-merdouble stranded DNA was passed over theprepared chips in a Biacore 2000 instru-ment and binding was seen only if theDNA contained G:G mismatches. DNAcontaining other SNPs or native DNA didnot bind to the ligand (Figure 1). Thesensitivity of the assay was shown by alinear increase in response using DNAconcentrations from 125 nM to 1000 nM,

a range that was optimised by changes insalt concentration. The intensity ofresponse was a function of both DNAconcentration and length.

Other non-gel based mismatch screeningmethods like melting curve analysis orMutS capture of mismatched fragmentsrely on detection systems such as labellingwith a fluorescent dye or the use oflabelled primers. Nakatani et al show thatBiacore’s SPR technology is sensitive,specific, requires no primers or probes andcan be adapted for high throughputanalysis (2).

This is the first report in which Biacore’sSPR technology has been used for DNAmutation analysis and while the design andsynthesis of ligands that are specific forother SNPs will be difficult, the principleof Nakatani’s work is a promisingdevelopment in accelerating DNAmutation analysis.

References1 Nakatani, K., Sando, S. and Saito, I.Scanning of guanine-guanine mismatches inDNA by synthetic ligands using surfaceplasmon resonanceNature Biotechnology 19:51-55 (2001)

2 Kwok, P.-Y. Reflections on a DNA mutationscanning tool Nature Biotechnology 19:18-19 (2001)

Figure 1. Sensorgram of binding of

mutant and native 27-mer

DNA to immobilized naphthyridine.

Duplexes containing native (G:C)

or mutated base pairings (T, A or

C) were injected for 180 seconds.