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DNA origami-based shape IDs for single-molecule nanomechanical genotyping

View Article: PubMed Central - PubMed

ABSTRACT

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

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Specificity of origami shape IDs.(a) Schematic showing of AuNP-assisted allele-specific primer extension of phiX 174 by a fully complementary primer (CP) or no extension by a mismatched primer (MP), the single mismatched base of which is at the 3′-end. (b) Agarose gel electrophoresis validated that CP could initiate the extension of template ssDNA into dsDNA, whereas MP could not. M, DL 15, 000 marker. (c) Upper: schematic showing of shape ID-based haplotyping. The site 1,433 is labelled with two M-strands with or without a single-base mismatch, which correspond to a triangular- and a cross-shaped ID, respectively. The site 4,914 is hybridized with a biotin-modified primer, which serves as the reference point. Lower: AFM images show that phiX 174 is correctly labelled with cross-shaped IDs (marked by tick), wrongly labelled with triangular (marked by cross), or with no labelling (marked by circle). The site 4,914 labelled with STV is marked with an arrowhead. The yield of specific labelling of shape IDs is ∼88%. Scale bar, 100 nm.
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f4: Specificity of origami shape IDs.(a) Schematic showing of AuNP-assisted allele-specific primer extension of phiX 174 by a fully complementary primer (CP) or no extension by a mismatched primer (MP), the single mismatched base of which is at the 3′-end. (b) Agarose gel electrophoresis validated that CP could initiate the extension of template ssDNA into dsDNA, whereas MP could not. M, DL 15, 000 marker. (c) Upper: schematic showing of shape ID-based haplotyping. The site 1,433 is labelled with two M-strands with or without a single-base mismatch, which correspond to a triangular- and a cross-shaped ID, respectively. The site 4,914 is hybridized with a biotin-modified primer, which serves as the reference point. Lower: AFM images show that phiX 174 is correctly labelled with cross-shaped IDs (marked by tick), wrongly labelled with triangular (marked by cross), or with no labelling (marked by circle). The site 4,914 labelled with STV is marked with an arrowhead. The yield of specific labelling of shape IDs is ∼88%. Scale bar, 100 nm.

Mentions: We further performed genotyping of SNPs, which forms the basis for many types of genetic analysis. Our previous studies showed that gold nanoparticles (AuNPs) could effectively inhibit mismatched binding3031. Hence, we employed AuNPs to assist discrimination of single-base mismatches in primer extension. To mimic polymorphic sites, we designed two primers with or without a single-base mismatch site at the 3′-end (Fig. 4a). Gel electrophoresis revealed that only the fully complementary primer could initiate primer extension to form the dsDNA circle, whereas the extension of single-base mismatched one was completely inhibited in the presence of AuNPs (Fig. 4b and Supplementary Fig. 23). To substantiate the specificity in AFM imaging, we chose the site 1,433 on phiX 174 to test two primers with or without a single-base mismatch, which correspond to a triangular and a cross shape ID, respectively (Fig. 4c). The site 4,914 was hybridized with a biotin-modified primer that could bind to STV, which served as the reference point. Statistical analysis of AFM images revealed that 88.0% of the phiX 174 template was correctly lableled with the cross shape ID, which confirms the high stringency of AuNPs-enhanced primer extension for SNP genotyping using shape IDs (Supplementary Fig. 25)31.


DNA origami-based shape IDs for single-molecule nanomechanical genotyping
Specificity of origami shape IDs.(a) Schematic showing of AuNP-assisted allele-specific primer extension of phiX 174 by a fully complementary primer (CP) or no extension by a mismatched primer (MP), the single mismatched base of which is at the 3′-end. (b) Agarose gel electrophoresis validated that CP could initiate the extension of template ssDNA into dsDNA, whereas MP could not. M, DL 15, 000 marker. (c) Upper: schematic showing of shape ID-based haplotyping. The site 1,433 is labelled with two M-strands with or without a single-base mismatch, which correspond to a triangular- and a cross-shaped ID, respectively. The site 4,914 is hybridized with a biotin-modified primer, which serves as the reference point. Lower: AFM images show that phiX 174 is correctly labelled with cross-shaped IDs (marked by tick), wrongly labelled with triangular (marked by cross), or with no labelling (marked by circle). The site 4,914 labelled with STV is marked with an arrowhead. The yield of specific labelling of shape IDs is ∼88%. Scale bar, 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384221&req=5

f4: Specificity of origami shape IDs.(a) Schematic showing of AuNP-assisted allele-specific primer extension of phiX 174 by a fully complementary primer (CP) or no extension by a mismatched primer (MP), the single mismatched base of which is at the 3′-end. (b) Agarose gel electrophoresis validated that CP could initiate the extension of template ssDNA into dsDNA, whereas MP could not. M, DL 15, 000 marker. (c) Upper: schematic showing of shape ID-based haplotyping. The site 1,433 is labelled with two M-strands with or without a single-base mismatch, which correspond to a triangular- and a cross-shaped ID, respectively. The site 4,914 is hybridized with a biotin-modified primer, which serves as the reference point. Lower: AFM images show that phiX 174 is correctly labelled with cross-shaped IDs (marked by tick), wrongly labelled with triangular (marked by cross), or with no labelling (marked by circle). The site 4,914 labelled with STV is marked with an arrowhead. The yield of specific labelling of shape IDs is ∼88%. Scale bar, 100 nm.
Mentions: We further performed genotyping of SNPs, which forms the basis for many types of genetic analysis. Our previous studies showed that gold nanoparticles (AuNPs) could effectively inhibit mismatched binding3031. Hence, we employed AuNPs to assist discrimination of single-base mismatches in primer extension. To mimic polymorphic sites, we designed two primers with or without a single-base mismatch site at the 3′-end (Fig. 4a). Gel electrophoresis revealed that only the fully complementary primer could initiate primer extension to form the dsDNA circle, whereas the extension of single-base mismatched one was completely inhibited in the presence of AuNPs (Fig. 4b and Supplementary Fig. 23). To substantiate the specificity in AFM imaging, we chose the site 1,433 on phiX 174 to test two primers with or without a single-base mismatch, which correspond to a triangular and a cross shape ID, respectively (Fig. 4c). The site 4,914 was hybridized with a biotin-modified primer that could bind to STV, which served as the reference point. Statistical analysis of AFM images revealed that 88.0% of the phiX 174 template was correctly lableled with the cross shape ID, which confirms the high stringency of AuNPs-enhanced primer extension for SNP genotyping using shape IDs (Supplementary Fig. 25)31.

View Article: PubMed Central - PubMed

ABSTRACT

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

No MeSH data available.


Related in: MedlinePlus