Nanomed

Genotype-Haplotype Microscopy (GHM).

GHM project can be divided in three different research branches:

Combined determination of genotype and haplotype by using probes which are detectable on single DNA molecule at a distance as close as 10 nucleotides.

The chromosomes in human cells occur in pairs (with the exception of the sex cells). One member of each chromosome pair is inherited from a person's father; the other member of the pair is inherited from that person's mother. But chromosomes do not pass from each generation to the next one as identical copies. Rather, the chromosome pairs undergo a process known as recombination. The members of each chromosome pair come together and exchange pieces. The result is a hybrid chromosome containing pieces from both members of a chromosome pair, and this hybrid chromosome is passed on to the next generation. Over the course of many generations, segments of the ancestral chromosomes are through repeated recombination events. Some of the segments of the ancestral chromosomes occur as regions of DNA sequences that are shared by multiple individuals (Figure 1).

Figure 1. The two ancestral chromosomes being scrambled through recombination over many generations to yield different descendant chromosomes. If a genetic variant marked by the A on the ancestral chromosome increases the risk of a particular disease, the two individuals in the current generation who inherit that part of the ancestral chromosome will be at increased risk. Adjacent to the variant marked by the A are many SNPs that can be used to identify the location of the variant.

These segments are regions of chromosomes that have not been broken up by recombination, and they are separated by places where recombination has occurred. These segments are the haplotypes that enable geneticists to search for genes involved in diseases and other medically important traits.
The most common form of variation in DNA sequence is the single-nucleotide polymorphism (SNP); SNPs are present at a frequency of about 1 in 1,000 nucleotides in humans.
We are developing a method to haplotyping by direct visualization of polymorphic sites on DNA molecules. Our approach utilizes atomic force microscopy to detect directly the polymorphic sites in DNA fragments. In principle, the methods of DNA sequencing give us information on the presence of SNPs along the DNA strand, that can be used to assemble all the combinations of SNPs that identify the various haplotypes, choosing an appropriate DNA fragment as standard and hybridizing it with DNA under investigation, we obtain DNA-heteroduplex strands (Figure 2).

Figure 2. Heteroduplexes-DNA formation. The heteroduplexes are characterized by the presence of one o more mismatches, due to the presence of different nucleotides. We use a protein mismatch repairing to mark the mismatch present in such DNA-heteroduplex molecules.

We are evaluating various approaches to detect the mismatch position along DNA stands. Firstly, we have investigated heteroduplexes of DNA with only one mismatch. Presently we are investigating heteroduplexes DNA molecules with two mismatches.

Construction of a genotype-haplotype microscope (GHM).

Simultaneously, to the haplotype determination, we are developing a scanning probe microscope (Figure3), based on AFM technology, which will be dedicated to investigate the DNA haplotype, the protein-protein interactions and the interactions between DNA and proteins.

Figure 3.CAD project of the GHM (middle), head of the microscope (left) and a scheme of the optical beam deflection technique used in GHM setup (right)..

We are building a prototype of the GHM using Physik Instrumente (PI) components (controller, translators, actuators and stages). Tests of cantilever thermal noise have given better results than those characteristic of commercial AFM (Figure 4).

Figure 4. Frequency spectra of cantilever Brownian motion measured in air.

With this prototype we have acquired various force-distance curves on Si surfaces, which are perfectly reproducible and show the well known regions of the force-distance curves (Figure 5).

Figure 5. Force-distance curves recorded by GHM prototype: unfiltered signal (left) and filtered signal (right).

Study of protein-protein and dna-protein interaction with GHM microscope.

The design and realization of protein nano-arrays aim to develop devices able to detect protein markers in very small and dilute samples, in which the protein markers concentration reaches values of the order of 10-16 M. Scanning probe microscopy is a powerful tool in studies that involve biological systems, our aim is to use GHM microscope to study the ligand-receptor interaction. The critical point with atomic force microscopy techniques is the roughness of the substrate surface: if the roughness of the surface is too high, it is impossible to recognize the protein bound to the surface. Therefore, we have looked for an appropriate method to functionalize the substrate surface. The first step is a formation of a layer of 3-aminopropyltriethoxysilane (APTES) on freshly cleaved mica surface. Figure 6 shows an AFM image of the mica-APTES surface with a low roughness.

Figure 6: AFM Tapping mode image of mica-APTES surface, that shows a very low roughness

After the APTES functionalization we have added another layer of a linker substance* able to bind properly Fc portion of the antibody and to leave the antigen binding sites in the antibody Fab portion accessible (Figure 7).

Figure 7: Scheme of the antibody immobilization on the mica-APTES-linker surface.

The roughness of the mica-APTES-linker surface is nearly unchanged, so that it is possible to study the antibody-antigen interaction by scanning probe microscopy.

Vincenzo Ierardi is a staff researcher, responsible of the GHM project. The team is directed by Professor Ugo Valbusa of the Physics Department of the University of Genova.
The project directly involves:
" Dr. Francesca Giacopelli and Prof. Roberto Ravazzolo of the Gaslini Institute of Genova for the studies on the DNA haplotype,
" Dr. Paolo Domenichini of the BiuBi s.r.l. Genova for the realization of GHM microscope,
" Dr. Francesca Ferrera and Prof. Gilberto Filaci of the CEBR-Center of Excellence for Biomedical Research of the University of Genova for the studies on ligand-receptor complexes.

* F. Ferrera, V. Ierardi, E. Millo, G. Filaci, G. Damonte, F. Indiveri, U Valbusa, "Development of a new procedure for controlled antibodies immobilization useful for the construction of an antibody nanoarray allowing the measurement of antigen-antibody immune-complex via label free technique", Italian Patent, 17/11/2009;




	Genotype-Haplotype Microscopy (GHM). GHM project can be divided in three different research branches: Combined determination of genotype and haplotype by using probes which are detectable on single DNA molecule at a distance as close as 10 nucleotides. The chromosomes in human cells occur in pairs (with the exception of the sex cells). One member of each chromosome pair is inherited from a person's father; the other member of the pair is inherited from that person's mother. But chromosomes do not pass from each generation to the next one as identical copies. Rather, the chromosome pairs undergo a process known as recombination. The members of each chromosome pair come together and exchange pieces. The result is a hybrid chromosome containing pieces from both members of a chromosome pair, and this hybrid chromosome is passed on to the next generation. Over the course of many generations, segments of the ancestral chromosomes are through repeated recombination events. Some of the segments of the ancestral chromosomes occur as regions of DNA sequences that are shared by multiple individuals (Figure 1). Figure 1. The two ancestral chromosomes being scrambled through recombination over many generations to yield different descendant chromosomes. If a genetic variant marked by the A on the ancestral chromosome increases the risk of a particular disease, the two individuals in the current generation who inherit that part of the ancestral chromosome will be at increased risk. Adjacent to the variant marked by the A are many SNPs that can be used to identify the location of the variant. These segments are regions of chromosomes that have not been broken up by recombination, and they are separated by places where recombination has occurred. These segments are the haplotypes that enable geneticists to search for genes involved in diseases and other medically important traits. The most common form of variation in DNA sequence is the single-nucleotide polymorphism (SNP); SNPs are present at a frequency of about 1 in 1,000 nucleotides in humans. We are developing a method to haplotyping by direct visualization of polymorphic sites on DNA molecules. Our approach utilizes atomic force microscopy to detect directly the polymorphic sites in DNA fragments. In principle, the methods of DNA sequencing give us information on the presence of SNPs along the DNA strand, that can be used to assemble all the combinations of SNPs that identify the various haplotypes, choosing an appropriate DNA fragment as standard and hybridizing it with DNA under investigation, we obtain DNA-heteroduplex strands (Figure 2). Figure 2. Heteroduplexes-DNA formation. The heteroduplexes are characterized by the presence of one o more mismatches, due to the presence of different nucleotides. We use a protein mismatch repairing to mark the mismatch present in such DNA-heteroduplex molecules. We are evaluating various approaches to detect the mismatch position along DNA stands. Firstly, we have investigated heteroduplexes of DNA with only one mismatch. Presently we are investigating heteroduplexes DNA molecules with two mismatches. Construction of a genotype-haplotype microscope (GHM). Simultaneously, to the haplotype determination, we are developing a scanning probe microscope (Figure3), based on AFM technology, which will be dedicated to investigate the DNA haplotype, the protein-protein interactions and the interactions between DNA and proteins. Figure 3.CAD project of the GHM (middle), head of the microscope (left) and a scheme of the optical beam deflection technique used in GHM setup (right).. We are building a prototype of the GHM using Physik Instrumente (PI) components (controller, translators, actuators and stages). Tests of cantilever thermal noise have given better results than those characteristic of commercial AFM (Figure 4). Figure 4. Frequency spectra of cantilever Brownian motion measured in air. With this prototype we have acquired various force-distance curves on Si surfaces, which are perfectly reproducible and show the well known regions of the force-distance curves (Figure 5). Figure 5. Force-distance curves recorded by GHM prototype: unfiltered signal (left) and filtered signal (right). Study of protein-protein and dna-protein interaction with GHM microscope. The design and realization of protein nano-arrays aim to develop devices able to detect protein markers in very small and dilute samples, in which the protein markers concentration reaches values of the order of 10-16 M. Scanning probe microscopy is a powerful tool in studies that involve biological systems, our aim is to use GHM microscope to study the ligand-receptor interaction. The critical point with atomic force microscopy techniques is the roughness of the substrate surface: if the roughness of the surface is too high, it is impossible to recognize the protein bound to the surface. Therefore, we have looked for an appropriate method to functionalize the substrate surface. The first step is a formation of a layer of 3-aminopropyltriethoxysilane (APTES) on freshly cleaved mica surface. Figure 6 shows an AFM image of the mica-APTES surface with a low roughness. Figure 6: AFM Tapping mode image of mica-APTES surface, that shows a very low roughness After the APTES functionalization we have added another layer of a linker substance* able to bind properly Fc portion of the antibody and to leave the antigen binding sites in the antibody Fab portion accessible (Figure 7). Figure 7: Scheme of the antibody immobilization on the mica-APTES-linker surface. The roughness of the mica-APTES-linker surface is nearly unchanged, so that it is possible to study the antibody-antigen interaction by scanning probe microscopy. "WHO'S WHO" OF THE PROJECT Vincenzo Ierardi is a staff researcher, responsible of the GHM project. The team is directed by Professor Ugo Valbusa of the Physics Department of the University of Genova. The project directly involves: " Dr. Francesca Giacopelli and Prof. Roberto Ravazzolo of the Gaslini Institute of Genova for the studies on the DNA haplotype, " Dr. Paolo Domenichini of the BiuBi s.r.l. Genova for the realization of GHM microscope, " Dr. Francesca Ferrera and Prof. Gilberto Filaci of the CEBR-Center of Excellence for Biomedical Research of the University of Genova for the studies on ligand-receptor complexes. * F. Ferrera, V. Ierardi, E. Millo, G. Filaci, G. Damonte, F. Indiveri, U Valbusa, "Development of a new procedure for controlled antibodies immobilization useful for the construction of an antibody nanoarray allowing the measurement of antigen-antibody immune-complex via label free technique", Italian Patent, 17/11/2009;

How to reach us Last update 6/23/10