Keeping Up With the Next Generation

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Presentation transcript:

Keeping Up With the Next Generation John R. ten Bosch, Ph.D., Wayne W. Grody, M.D., Ph.D. The Journal of Molecular Diagnostics Volume 10, Issue 6, Pages 484-492 (November 2008) DOI: 10.2353/jmoldx.2008.080027 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Genome Enrichment Strategies. A: Megaplex PCR. Surface bound primers are extended in the first and second PCR cycle. Additional cycles are performed using universal primers in solution. U1 and U2 (green) are universal primer sequences. F and R (red) are unique primer sequences. B: Molecular inversion probe circularization. The 5′ and 3′ ends (red) of the MIP probe (black) hybridize to complementary genomic DNA, leaving a 1-bp gap that is filled in by DNA ligase. U1 and U2 (green) are universal primer sequences within the MIP probe. C: Connector inversion probe circularization. The 5′ and 3′ ends (red) of the CIPer probe (black) hybridize to complementary genomic DNA (gray), leaving a sizeable gap that is extended by DNA polymerase and ligated to the 3′ arm with DNA ligase. U1 and U2 (green) are universal primer sequences within the CIPer probe. D: Selector probe circularization. Genomic DNA (gray) is fragmented with a restriction endonuclease. Selector probes (black) with single-stranded overhangs (red) are ligated to the genomic restriction fragment ends (red) complementary to the selector probe overhangs. U1 and U2 (green) are universal primer sequences within the selector probe. E: Gene-collector circularization. A multiplex PCR is performed generating amplicons with unique sequence ends. Gene-collector probes (black) with single-stranded overhangs (red) are ligated to single-stranded overhangs (red) complementary to ends generated from the PCR reaction. F: Microarray pull-down assay. Genomic DNA (gray, with regions of interest in red, blue, and purple) is randomly sheared then ligated to universal adapter sequences (green). Adapter-ligated DNA is then hybridized to the array, eluted, and amplified with universal primers. G: cDNA sequencing. mRNA is isolated, converted to cDNA, sheared, ligated with adapters (green), and sequenced. The Journal of Molecular Diagnostics 2008 10, 484-492DOI: (10.2353/jmoldx.2008.080027) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions