1. Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays 
Janice M. Spence, Paul G. Rothberg, Nancy Wang, W. Richard Burack 
The Journal of Molecular Diagnostics 
Volume 13, Issue 3, Pages (May 2011)
DOI: /j.jmoldx
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Map of primer locations and feature sites of the BCL2 region. This schematic representation of 35 kb of human chromosome 18q21 shows the distribution of primers used for MPAD analysis (pool A, upward long black lines; and pool B, upward short gray lines) and BIOMED-2 (tubes A, B, and C; downward black lines). DNA regions corresponding to the features on the LDA are represented by gray bars. The scale refers to coordinates of chromosome 18q21 according to National Center for Biotechnology Information build 37.1 ( last accessed October 26, 2010).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Representative MPAD assay. PCR products are hybridized to the LDA containing the designated BCL2 features, placed in duplicate. After high-stringency washes, the LDAs are incubated with streptavidin–horseradish peroxidase to label products containing the biotinylated IGH-JH and PAX9 primers and visualized with enhanced chemoluminescence. Feature placement is denoted by the numbered LDA (far right) and identified below. Specimens CHL1 and CHL2 are negative controls. Specimens 28 through 33 are FL. All samples show a positive reaction for the PAX9 PCR control region (feature 1). Specimens 28, 30, and 31 show the combined MBR and extended (Ext) MBR reaction pattern (features 2 and 3), often observed from translocations within the 150-bp MBR of the BCL2 region; specimens 29 and 33 show a single MBR reaction (feature 2), indicating a translocation site upstream of the canonical MBR site. Feature key: 1, PAX9 (PCR control); 2, MBR; 3, Ext MBR; 4, 3′MBR 1; 5, 3′MBR 2; 6, minor cluster region (mcr) 1; 7, mcr 2; 8, icr. Figure 1 is a graphic representation of each feature region on chromosome 18.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
The 2 × 2 contingency tables comparing MPAD, FISH, and BIOMED-2 translocation results from FL specimens. Each contingency table has a row appended with a comparison of the results with the gold standard of positive by any method.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Translocation sites within the BCL2 region. BCL2 is located on 18p21 and is orientated telomere to centromere. Twenty-two translocations sites were sequenced and mapped to the untranslated 3′ region of BCL2 exon 3 and beyond; all translocation sites identified in this study are centromeric to the translated BCL2 sequence. Individual break point sites are denoted by vertical lines. Of these sites, 14 (64%) were in the 150-bp MBR region (expanded top map), 3 (14%) were in the 3′MBR region (expanded lower map), 3 (14%) were in the icr region, and 2 (9%) were in the minor cluster region (mcr). Identical translocation sites were found in two specimens, which were later determined to be from patients experiencing a relapse of their FL, as detailed in Table 1. The scale refers to coordinates of chromosome 18, according to National Center for Biotechnology Information build 37.1 ( last accessed October 26, 2010).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Sensitivity of multiplex PCR amplification and product detection. To assess the sensitivity of the multiplex PCR, tumor DNA was diluted in BCL2 normal FFPE DNA at the designated ratios and used as template in PCRs with primer pools A and B. A: The PCR products (20 μL) from diluent control (dil) and tumor dilutions (1:4 to 1:500) were analyzed on a 2% agarose gel (NuSieve), stained with ethidium bromide, and photographed with UV transillumination. Pool A primers generated an approximate 350-bp nonspecific product, whereas pool B primers show a smattering of small products. This illustrates the difficulty of interpreting highly multiplexed PCR by gel electrophoresis. The arrow (lane 9) denotes the specific amplicon from this tumor specimen. This band is not distinguishable at a template dilution greater than 1:4 in BCL2 normal DNA. B: The LDA results from these same PCR products. The positive signal persists through the 1:20 dilution and is still visible at a 1:100 dilution of tumor DNA.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions