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

Isolate DNA

PCR with primers that flank theregion to be examined

Hybridize with ASO that iscomplementary to normal allele

Hybridize with ASO that iscomplementary to variant allele

CG T

Hybridization No hybridization

G

AT

A

No hybridization hybridization

C

AT

CG

CG

ATC

+

+

C A

Normal allele Variant allele

Genomic DNA

PCR-amplified DNA

(split the sample,isolate the PCR products by electtrophoresisand blot.)

Blood sample from a cystic fibrosis carrier

PCR product

Person's genotype: N/N N/A A/A(N: normal allele; A. affected allele) (normal) (carrier) (affected)

Hybridization with normal ASO

N/N N/A A/A(normal) (carrier) (affected)

Hybridization with variant ASO

Figure 3.7b. ASO-based detection of variant alleles: results.. Blood samples from three individualsanalyzed by ASO hybridization as described in figure 3.7a. The homozygous normal individual showshybridization only with the normal ASO, the heterozygous individual shows hybridization with bothASOs 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.

Normal Cells Tumor cells

mRNA mRNA

red cDNA green cDNA Mix and hybridize with DNA array Results:

Red spots: Yellow spots: Green spots: genes that are (most of them) genes that are under expressed equally expressed over expressed in the tumor. genes. in the tumor.

Biopsy

Figure 1The same section of the microarray is shown for three independent hybridizations comparing RNA isolated at the 8hour time-point after serum treatment to RNA from serum-deprived cells. Each microarray contained 9996elements, including 9804 human cDNAs, representing 8613 different genes. mRNA from serum-deprived cells wasused to prepare cDNA labeled with Cy3-dUTP and mRNA harvested from cells at different times after serumstimulation was used to prepare cDNA labeled with Cy5-dUTP. The two cDNA probes were mixed andsimultaneously hybridized to the microarray. The image of the subsequent scan shows genes whose mRNAs aremore abundant in the serum-deprived fibroblasts (i.e., suppressed by serum treatment) as green spots and geneswhose mRNAs are more abundant in the serum treated fibroblasts appear as red spots. Yellow spots represent geneswhose expression does not vary significantly between the two samples. The arrows indicate the spots representingthe following genes: 1) Protein disulphide isomerase-related protein P5, 2) Interleukin-8 precursor, 3) ESTAA057170, 4) Vascular endothelial growth factor.

Protein-based ToolsWestern 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 ToolsImmuno-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 ToolsEnzyme 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

Nucleic acid Basics

Hybridization

Electrophoresis

PCRDiagnostic

tools

DNA-Proteininteractions

Chromatin

Gene expression

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

HHydrogen bond acceptor H Hydrogen bond donor VdW VanderWaals interaction

Note the positions of the glycosidic bonds with respect to the major and minor grooves

H

H H

H

H

H

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