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PCR amplification of DNA containing each of nine different microsatellites from each progeny plant with one of each locus-specific primer pairs, analysis of product mixtures by capillary electrophoresis, detection of fluorescent products, and generation of electropherograms were as described under “Materials and Methods.” A, Electropherograms showing all PCR products; one of each primer pair was labeled with FAM or HEX fluorescent dye. B, Representative electropherogram patterns for locus NGA1107. Apparent absolute size of primary wild-type (wt) product band is 317 bp. Shown also are products where both alleles showed an increase of one repeat unit, increasing apparent product length to 319 bp (+2), where one allele maintained wild-type length and one showed a loss of one repeat [wt & (-2)], and where one allele remained wild type and one showed a shift of two repeats, decreasing apparent length to 313 bp [wt & (-4)]. Arrows indicate position of 317-bp (wild-type) product. C, Patterns for reconstruction mixtures. DNA from plants homozygous (pure) for alleles at locus NGA1107 encoding 313-bp (black arrow) or 315-bp (gray arrow) products or mixtures of the two at indicated ratios (concentrations of DNAs measured before PCR by staining with PicoGreen [Molecular Probes, Eugene, OR]) were analyzed as described above. Note that the primary criterion for scoring a pattern as reflecting a mixture of two different length products—second peak from right greater than a significantly large first peak—is met only for 1:1 and 2:1 mixtures.

Journal:

Article Title: Reduction of Stability of Arabidopsis Genomic and Transgenic DNA-Repeat Sequences (Microsatellites) by Inactivation of AtMSH2 Mismatch-Repair Function 1

doi: 10.1104/pp.103.023952

Figure Lengend Snippet: PCR amplification of DNA containing each of nine different microsatellites from each progeny plant with one of each locus-specific primer pairs, analysis of product mixtures by capillary electrophoresis, detection of fluorescent products, and generation of electropherograms were as described under “Materials and Methods.” A, Electropherograms showing all PCR products; one of each primer pair was labeled with FAM or HEX fluorescent dye. B, Representative electropherogram patterns for locus NGA1107. Apparent absolute size of primary wild-type (wt) product band is 317 bp. Shown also are products where both alleles showed an increase of one repeat unit, increasing apparent product length to 319 bp (+2), where one allele maintained wild-type length and one showed a loss of one repeat [wt & (-2)], and where one allele remained wild type and one showed a shift of two repeats, decreasing apparent length to 313 bp [wt & (-4)]. Arrows indicate position of 317-bp (wild-type) product. C, Patterns for reconstruction mixtures. DNA from plants homozygous (pure) for alleles at locus NGA1107 encoding 313-bp (black arrow) or 315-bp (gray arrow) products or mixtures of the two at indicated ratios (concentrations of DNAs measured before PCR by staining with PicoGreen [Molecular Probes, Eugene, OR]) were analyzed as described above. Note that the primary criterion for scoring a pattern as reflecting a mixture of two different length products—second peak from right greater than a significantly large first peak—is met only for 1:1 and 2:1 mixtures.

Article Snippet: Up to nine locus-specific PCR reaction products were diluted as empirically found appropriate for subsequent analysis, mixed, and analyzed by capillary electrophoresis and measurement of DNA-band fluorescent intensities, using the ABI Prism 3100 Genetic Analyzer and associated software (Applied Biosystems, Palo Alto, CA).

Techniques: Amplification, Electrophoresis, Labeling, Staining