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Comment
. 2013 May;51(5):e79-83.
doi: 10.1515/cclm-2012-0429.

Comparison of biological specimens and DNA collection methods for PCR amplification and microarray analysis

Jasmine A Rethmeyer, Xiaoyu Tan, Ann Manzardo, Stephen R Schroeder, Merlin G Butler
Comment

Comparison of biological specimens and DNA collection methods for PCR amplification and microarray analysis

Jasmine A Rethmeyer et al. Clin Chem Lab Med. 2013 May.
No abstract available

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Figures

Figure 1
Figure 1
Electrophoretic analysis of genomic DNA from buccal cell and saliva samples showing mixed results with DNA (100–200 ng) loaded onto 1% agarose gel and visualized using 0.5 μg/mL of ethidium bromide. (A) Stored buccal cell DNA isolated with the QIAamp (Qiagen) kit from nine infants with developmental delay in which seven DNA samples (Subjects 52, 102, 120, 144, 165, 188, 223) met standardized laboratory criteria for chromosomal microarray analysis based on DNA quantity or yield (e.g., 0.75 μg), purity or qualtiy (i.e., spectrophotometer OD 260/280 ratios; e.g., 1.6–2.1) and sufficient intact high molecular weight DNA using gel electrophoresis. (B) Stored buccal cell DNA isolated with the QIAamp (Qiagen) kit from three representative infants (Subjects 95, 100, 175) with developmental delay showing the degree of degradation from 10,000 bp to 1000 bp range as designated by known DNA markers and not meeting laboratory criteria whereas a greater yield of high quality intact DNA was found with MasterPure DNA kit in five representative infants (Subjects 106, 112, 117, 122, 135). No intact DNA fragments were visualized below 1000 bp. (C) Chromosomal microarray analysis of buccal DNA from Subject 165 using the Affymetrix Genome-Wide Human SNP Array 6.0 (Santa Clara, CA, USA) to identify genomic deletions or duplications showed a 7 Mb deletion (copy number of 1) of the 20q13.2–20q13.33 region occurring at 53,512,484–60,850,110 bp from the p-terminus of the chromosome. (D) DNA isolated from freshly-collected buccal and saliva using the QIAamp (Qiagen) kit from four control subjects meeting laboratory criteria except for one buccal sample (Subject 2) with a high OD ratio of 2.6 in comparison with DNA isolated from blood and lymphoblasts (L-blast), more conventional sources for DNA, obtained from two different representative control subjects (Subjects 1046, 1047) showing the typical DNA pattern with gel electrophoresis ranging from 10,000 bp to 1000 bp designated by known DNA markers. No intact DNA fragments were visualized below 1000 bp.
Figure 2
Figure 2
PCR amplification of the growth hormone receptor (GHR) gene with DNA isolated from both plasma and buccal cells. Lanes 43 and 44 show two PCR fragments representing heterozygosity of the GHR gene from Subject 1 using fresh plasma DNA and fresh buccal DNA, respectively; Lane 45 shows a single PCR fragment representing homozygosity using fresh buccal DNA from Subject 2, and Lanes 46–48 represent unsuccessful PCR amplification using stored frozen plasma DNA from Subjects 3, 4, and 5.
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