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For mCGH (12), we reamplified and purified the initial iWGA products.
DNA fingerprinting of iWGA aliquots was performed with nonstained HT-29 cells at the level of 10 pooled cells and 5 pooled cells (see Table 1 in the online Data Supplement).
The DNA fingerprint analysis of the respective iWGA aliquots yielded a mean PCR performance of 87.4% (range, 58%-100%).
The heterozygous signals were assessed quantitatively, and the signal ratios ranged from subheterozygous (wild type-mutant ratio, 1:4) to superheterozygous (wild type-mutant ratio, 5:1) owing to preferential amplification in the course of iWGA. The ADO rates were similar across the reactions at all levels [ranging between 56% (5 of 9 loci) and 67% (6 of 9 and 12 of 16 loci, respectively], yielding PCR performances of 72% (10-cell level), 67% (5-cell level), and 63% (single-cell level) (see Table 3 in the online Data Supplement).
In 2 additional approaches, we pooled the iWGA products of 5 single cells and the products of 3 single cells (Table 2; see Table 3 in the online Data Supplement) after DNA fingerprint analysis revealed the cells to be of HT-29 origin.
To check the concordance between the CGH profiles, we compared chromosomal CGH data of iWGA aliquots from 3 single cells double-positive for cytokeratin and TO-PRO-3 with non-preamplified DNA isolated from cultured HT-29 cells (bulk HT-29 genomic DNA) (Fig.
Our implementation of an iWGA step between the laser microdissection of cells and DNA fingerprinting created the potential to perform multiple analyses from a particular cell.
One purpose of DNA fingerprint analysis of iWGA products was to assess the quality of the DNA amplified from single cells.
The iWGA method we used leads to comparatively less-effective amplification.
Although a complete (heterozygous) SNP profile of HT-29 cells could be obtained by summarizing the data from 2 to 4 single cells, sequencing of pooled iWGA samples did not produce a full SNP "profile." The reason for this effect is not clear.
Using reference DNA from iWGA results for pooled microdissected cells, we obtained interpretable profiles for all 3 iWGA samples from single cells.
Some aberrations may be attributed to inherent problems of iWGA with rare cells.
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