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NEB 2 log ladder was used. See Figure A12-5 legend for template designations. Slight band in lane Zn was observed inconsistently and infrequently and is attributed to contamination during nested PCR.

Correct size of amplified TCRα and β products after nested PCR. Correct size is around 800 bp. Intrinsically the TCRβ chain is larger than the TCRα chain; however, due to the design of RACE primers, the TCRα chain fragment is slightly larger after nested PCR

Two rounds of nested PCR amplifications are used in which one or two poison primers are added to the first-round PCR mix (top schematic). In the first round reaction, full-length and poison amplicons are produced (middle schematic). During the second round of amplification in which the poison primers are not added, the wild-type and deletion amplicons are produced (lower schematic). Addition of the poison primers in the first round reaction reduces the amount of full-length amplicon produced, thereby allowing the slightly smaller deleted amplicons to outcompete the amplification of the wild-type amplicon in the second round of PCR.

Workflow of an exon skipping evaluation experiment. RNA evaluation: a 6-well plate is seeded and transfected with two different AON concentrations. Cells are collected after 48 h for RNA extraction, reverse transcription (RT), and nested PCR analysis by densitometry. Dystrophin protein quantification: a 96-well plate is seeded, transfected, and allowed to differentiate for a week. Plates are fixed before being analyzed by myoblot . Created with Biorender.com

Detection of SVV DNA in ganglia from monkeys naturally infected with SVV. DNA extracted from pooled cervical (C), thoracic (T), lumbar (L), and sacral (S) ganglia, lung and liver from two monkeys (natural-1 and -2) exposed to an intratracheally infected monkey (intratracheal) was analyzed by nested PCR using primers and probes specific for SVV ORF 63. DNA was omitted in one of the reactions (No DNA). DNA from uninfected (BSC-1 DNA) and SVV-infected BSC-1 cells in culture (SVV-BSC-1 DNA) was used as a negative and positive control, respectively. (Modified and reprinted with permission of J. Virol.)

Nested PCR results. (a) Skipped and non-skipped product schematic representations. Primers 48F and 54R were used in the first PCR and primers 49F and 53R in the second. Skipped product size is reduced due to the lack of exon 51 and 52 while the non-skipped product is just lacking exon 52. Created with Biorender.com. (b) Agarose gel image from AON transfection. Fifty microliter of PCR product were examined in a 2% agarose gel stained with SYBRSafe. Image was captured in a Gel Doc™ EZ Imager (BIORAD). Red boxes indicate the quantified bands. The described relative percentage of exon skipping for 100 nM AON was 95.41% and for 300 nM AON was 53.62%

Detection of SVV ORF 63-specific DNA in peripheral blood MNC subpopulations of SVV-infected adult African green monkeys. MNCs from an uninfected monkey and two SVV-infected monkeys (M7 and M8) at 14 months (a) and 23 months (b) after intratracheal inoculation with SVV were sorted by flow cytometry using (a) anti-CD4, anti-CD14 and anti-CD20 monoclonal antibodies and (b) anti-CD4 and anti-CD8 monoclonal antibodies. MNC DNA from the acutely infected monkey (M1) was used as a positive control. DNA extracted from the MNC populations was analyzed by nested PCR followed by Southern blot hybridization. SVV DNA was detected exclusively in CD4₊ and CD8₊ cells. (Modified and reprinted with permission of Virology.)