Journal: Nucleic Acids Research
Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
Figure Lengend Snippet: Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.
Article Snippet: MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8).
Techniques: Amplification, Blocking Assay, Generated, Magnetic Beads, Polymerase Chain Reaction