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DNA-based Tools • ASO (Allele Specific Oligonucleotides) – Used to detect specific alleles. – Can determine if a person is homozygous or heterozygous for a particular allele Blood sample from a cystic fibrosis carrier Isolate DNA Normal allele Variant allele C G + C G A T C PCR with primers that flank the region to be examined A T + Genomic DNA PCR-amplified DNA (split the sample, isolate the PCR products by electtrophoresis and blot.) Hybridize with ASO that is complementary to normal allele C C G T A A T G C Hybridization Hybridize with ASO that is complementary to variant allele No hybridization A No hybridization hybridization Person's genotype: (N: normal allele; A. affected allele) N/N N/A A/A (normal) (carrier) (affected) N/N N/A A/A (normal) (carrier) (affected) PCR product Hybridization with normal ASO Hybridization with variant ASO Figure 3.7b. ASO-based detection of variant alleles: results.. Blood samples from three individuals analyzed by ASO hybridization as described in figure 3.7a. The homozygous normal individual shows hybridization only with the normal ASO, the heterozygous individual shows hybridization with both ASOs and the individual who is homozygous affected shows hybridization with only the variant ASO. DNA-based Tools • ASO (Allele Specific Oligonucleotides) – Good Knowledge of the allele to be tested is required for distinguishing one allele from another – Usually used in conjunction with PCR, electrophoresis and DNA blotting • Other technologies for detecting specific point mutations are available – All suffer from the disadvantage that each test is directed toward a specific allele. A person affected by a different variant allele will not be detected as being affected. DNA-based Tools DNA Sequencing • Good for: – Idnetifying uncommon mutations • Disadvantages – Phenotype may not be apparent from genotype – Mutations in promotor or introns that affect gene expression may be missed DNA-based Tools DNA Chips • Primarily a research tool for discovery of disease genes. Biopsy Normal Cells Tumor cells mRNA mRNA red cDNA green cDNA Mix and hybridize with DNA array Results: Red spots: genes that are under expressed in the tumor. Yellow spots: (most of them) equally expressed genes. Green spots: genes that are over expressed in the tumor. Figure 1 The same section of the microarray is shown for three independent hybridizations comparing RNA isolated at the 8 hour time-point after serum treatment to RNA from serum-deprived cells. Each microarray contained 9996 elements, including 9804 human cDNAs, representing 8613 different genes. mRNA from serum-deprived cells was used to prepare cDNA labeled with Cy3-dUTP and mRNA harvested from cells at different times after serum stimulation was used to prepare cDNA labeled with Cy5-dUTP. The two cDNA probes were mixed and simultaneously hybridized to the microarray. The image of the subsequent scan shows genes whose mRNAs are more abundant in the serum-deprived fibroblasts (i.e., suppressed by serum treatment) as green spots and genes whose mRNAs are more abundant in the serum treated fibroblasts appear as red spots. Yellow spots represent genes whose expression does not vary significantly between the two samples. The arrows indicate the spots representing the following genes: 1) Protein disulphide isomerase-related protein P5, 2) Interleukin-8 precursor, 3) EST AA057170, 4) Vascular endothelial growth factor. Protein-based Tools Western Blots • Good for: – detecting the presence of a protein (e.g. Antitrypsin III) – detecting the presence of an antibody • Disadvantages: – Can not determine if protein is active or not (e.g. antitrypsin III) – Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required) Protein-based Tools Immuno-histochemistry • Good for: – Identifying tissues that express a particular protein – Showing over- or under- expression of a protein in a tissue (e.g. her-2 protein in breast cancer biopsy) • Disadvantages: – Can not determine if protein is active or not (e.g. antitrypsin III) – Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required) Protein-based Tools Enzyme Assay • Good for: – determining the activity of a suspect enzyme – determining the concentration of a metabolite • Disadvantages: – Must have access to tissue where protein is expressed (e.g. invasive biopsy might be required) – Protein must be an enzyme or influence an enzyme activity – Activity must be stable during isolation of tissue DNA-based Tools FISH (Fluorescence In Situ Hybridization) • Good for: – visualization of large chromosomal changes • Disadvantages: – Can not be used to detect small genetic changes DNA-based Tools PCR • Good for: – amplifying the amount of DNA. – isolating a segment of DNA • Disadvantages: – Only semiquantitative – Linited size of segment to be amplified DNA-based Tools PCR • Examples: – Diagnosis of infectious disease (e.g. clamydia, gonorrhea, HIV) – Detection of a small deletion or insertion (e.g. the ΔF508 cystic fibrosis allele) – Amplification of a specific region to obtain DNA for other tests (e.g. ASO testing) DNA-based Tools ASO (Allele Specific Oligonucleotides) •Good for: – Distinguishing between known alleles that have small differences in DNA sequence (e.g. point mutations, small deletions or insertions) •Disadvantages: – One needs to know the DNA sequences of the normal DNA and of the allele to be tested. DNA-based Tools DNA sequencing • Good for: – Idnetifying uncommon mutations • Disadvantages – Phenotype may not be apparent from genotype – Mutations in promotor or introns that affect gene expression may be missed DNA-based Tools DNA Chips • Currently a research tool for discovery of disease genes. • A potentially powerful diagnostic tool Hybridization Nucleic acid Basics PCR Electrophoresis DNA-Protein interactions Chromatin Gene expression Diagnostic tools Most Proteins bind in the Major Groove of DNA Major groove is visible here (minor groove would be seen from the back of the slide) Minor groove is visible here (major groove would be seen from the back of the slide) Important functional groups located in the major groove H Hydrogen bond acceptor H H H Hydrogen bond donor VdW VanderWaals interaction H H H H Note the positions of the glycosidic bonds with respect to the major and minor grooves Examples of Amino acid-Base Interactions Common DNA-binding motifs Fig. 16.6 from Mark’s Basic Medical Biochemistry (page 288) Common DNA-binding motifs Specific Examples The examples presented can be viewed via the “DNA-protein interactions” tutorial on the Molecular Basis of Medicine Web site