Sensitive Methods for Single Nucleotide Polymorphism Detection
Written by Hadar Nir
Hadar Nira, Vered Bronnerd, Monica Dinesd, Tsafrir Bravmand, Gadi Shusterb and Yoav Eichena,c
aDepartment of Chemistry, bDepartment of Biology, cSolid State InstituteTechnion – Israel Institute of Technology, Technion City 32000, Haifa Israel.d Bio-Rad Haifa, Haifa 32000, Israel.
Single Nucleotide Polymorphisms (SNPs) are the most common genetic mutations in the human genome and are an important source of genetic markers which help identify many hereditary diseases. The development of sensitive genetic analysis methods to detect these single base mutations requires high sensitivity, state-of-the-art selectivity, preferable combined with miniaturization and speed. In our study, we combine a selective enzymatic method with ultra sensitive Surface Plasmon Resonance (SPR) technologies (ProteOn XPR36), or with a simple and inexpensive detection technology like optical or electrical measurements. We use DNA polymerase enzyme in two different reaction methods and the amplification is accomplished by bio catalyzed precipitation. This approach allows the reliable and reproductive detection of as low as 0.5 amoles (~300,000 molecules) of SNPs with SPR technology.We also use enzymatic primer elongation with DNA polymerase on solid substrate to selectively incorporate biotin (biotin-11-dUTP) on the specific SNP location. By using biotin-streptavidine conjugation, we localize straptavidine-nanogold on the biotinylated DNA, which serve as nucleation center for further gold deposition. This procedure enhances the SPR signal, enabling us to detect lower number of DNA molecules. This gold spot can be measured also by electrical measurements and even by the naked eye.