Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples - PubMed (original) (raw)

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Gang Wang et al. Nucleic Acids Res. 2004.

Abstract

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.

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Figures

Figure 1

Figure 1

Protocol used for the unbiased amplification of two genomic DNAs via balanced-PCR.

Figure 2

Figure 2

Evaluation of the specificity of primers P2a and P2b for amplifying target and control genomes ligated to LN1 and LN2, respectively. The protocol of Figure 1 was applied to co-amplify and, subsequently, to re-separate the two genomes. Lane 1, P2a-amplified genome; lane 4, P2b-amplified genome; lanes 2 and 3, the products depicted in lanes 1 and 4 were further amplified for 10 cycles using P2b and P2a primers, respectively.

Figure 3

Figure 3

Reproducibility of array-CGH screening of samples amplified via balanced-PCR. In two independent experiments, genomic DNA from BT474 and HMEC cells was amplified via balanced-PCR and then screened on different human cDNA microarrays. Fold change versus chromosomal position for chromosomes 17 (481 genes) and 20 (218 genes) are depicted.

Figure 4

Figure 4

Array-CGH screening of genomic DNA from human female BT474 and HMEC cells, using unamplified DNA. Chromosomes 1–23 are depicted and arrows indicate regions of known amplifications and deletions for the BT474 cell line.

Figure 5

Figure 5

Array-CGH screening of genomic DNA from human female BT474 and HMEC cells, using balanced-PCR amplified DNA. Chromosomes 1–23 are depicted and arrows indicate highlighted regions of known amplifications and deletions for the BT474 cell line.

Figure 6

Figure 6

Array-CGH screening of chromosomes 17 and 20 from human female BT474 and HMEC cells: comparison of results using unamplified DNA (top curve), balanced-PCR amplified DNA (middle curve) and MDA amplified DNA (bottom curve).

Figure 7

Figure 7

Real-time PCR screening (TaqMan assay) of relative gene copy numbers for breast cancer cells (BT747, ‘target’) versus HMEC cells (‘control’). First column (black), amplification directly from unamplified genomic DNA. Second column (dark gray), amplification from balanced-PCR amplified genomic DNA. Third column (light gray), amplification from MDA amplified genomic DNA.

Figure 8

Figure 8

Screening of DNA from paraffin-embedded DNA. (A) Gel electrophoresis profile from a formalin-fixed, paraffin-embedded sample indicating DNA degradation. (B) Array-CGH screening of all 23 chromosomes using unamplified DNA (top curve), balanced-PCR-amplified DNA (middle curve) and MDA amplified DNA (bottom curve). (C) Chromosome 4 area of interest, indicating a 7 Mb amplification region in the unamplified and the balanced-PCR amplified sample. Duplicate experiments on two different arrays are depicted. (D) Evaluation of single genes using unamplified DNA, balanced-PCR amplified DNA and MDA-amplified DNA using Taqman real-time PCR.

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