Molecular Approaches for Screening of Genetic Diseases

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Molecular Approaches for Screening of Genetic Diseases Ali J. Marian, MD, FCCP CHEST Volume 108, Issue 1, Pages 255-265 (July 1995) DOI: 10.1378/chest.108.1.255 Copyright © 1995 The American College of Chest Physicians Terms and Conditions

Figure 1 A polymorphic STR marker. DNA obtained from normal (N) and affected (A) individuals in a family with hypertrophic cardiomyopathy was amplified by PCR. Radiolabeled primers were used in the PCR reaction. Gel electrophoresis and autoradiography showed the presence of five alleles (variations) for the marker in this family. They are labeled 1 through 5 on the autoradiogram. Allele 2 is present in all affected individuals and is absent in all normal individuals, showing cosegregation with inheritance of the disease. Such analysis is used in linkage studies. CHEST 1995 108, 255-265DOI: (10.1378/chest.108.1.255) Copyright © 1995 The American College of Chest Physicians Terms and Conditions

Figure 2 A simple diagram illustrating a linkage study. A polymorphic DNA marker (GT)n is present in each chromosome (allele). Four variations in the number of DNA repeats are shown in this small family. Allele 2 is the only allele that cosegregates with the inheritance of the disease. It is present in affected (full circle or squares) and is absent in normal individuals. A computer program is used to calculate the odds ratio of inheritance of an allele with the disease. A LOD score of greater 3 is considered significant. CHEST 1995 108, 255-265DOI: (10.1378/chest.108.1.255) Copyright © 1995 The American College of Chest Physicians Terms and Conditions

Figure 3 Diagram illustrating the technique of chemical mismatch cleavage. DNA (or cDNA) from normal and affected individuals is amplified in the region of interest by PCR. The PCR reaction of the normal DNA template contains radiolabeled primers to synthesize DNA probes. The PCR products are denatured and reannealed to form heteroduplexes. In the presence of a mutation, a mismatch occurs, which is modified by reaction with OsO4 and HA. The mismatched nucleotide is subsequently cleaved by piperidine. The products of cleavage are separated by gel electrophoresis and identified by autoradiography. CHEST 1995 108, 255-265DOI: (10.1378/chest.108.1.255) Copyright © 1995 The American College of Chest Physicians Terms and Conditions

Figure 4 Diagram showing the technique of RNase protection assay. DNA or cDNA from an affected individual in the region of interest is amplified by PCR and hybridized to a radiolabeled normal RNA probe. The RNA:DNA hybrid is then subjected to digestion with RNase A that cleaves the single-stranded RNA at the site of a mismatch. The digested products are separated by gel electrophoresis and are identified by autoradiography. Exact site and nature of the mutation are subsequently detected by sequencing. CHEST 1995 108, 255-265DOI: (10.1378/chest.108.1.255) Copyright © 1995 The American College of Chest Physicians Terms and Conditions