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Comparative Study
. 2004 Jan;164(1):23-33.
doi: 10.1016/S0002-9440(10)63092-1.

Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping

Martha S Rook  1 , Scott M Delach, Galina Deyneko, Andrew Worlock, Jia Liu Wolfe
Affiliations
Comparative Study

Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping

Martha S Rook et al. Am J Pathol. 2004 Jan.

Abstract

Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a ø29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied.

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Figures

Figure 1
Figure 1
A: Representative LCM images of a colon cancer cell microdissection. B: TaqMan SNP-genotyping assay plot for G/A1182 of the EDNRB gene with WGA products from 70 different 3000 prostate LCM cell caps and WGA products from 12 different 1500 colon LCM cell caps. Assays were done in replicates. Arbitrary fluorescence units are shown on axes.
Figure 2
Figure 2
Schematic sample preparation and data analysis.
Figure 3
Figure 3
Effect of lysis procedure on a TaqMan SNP-genotyping assay for C/G16996 of the ERBB3 gene. LCM colon cell caps containing 1500 cells (8 caps), 750 cells (12 caps), and 300 cells (8 caps) were lysed by a proteinase K (pk) or an alkaline (alk) lysis protocol. All samples should give a heterozygote call. No calls are highlighted by open circles and miscalls are highlighted by filled circles. Shown on axes are arbitrary fluorescence units.
Figure 4
Figure 4
A: Effect of pooling WGA products from a 300 or a 100 LCM prostate cell cap on TaqMan SNP-genotyping assay for A/G36177 of the CYP3A5 gene: 24 WGA products were assayed in replicates for each cell number to afford 96 unpooled data points; 8 individual WGA products were assayed in replicates for each cell number to afford 32 pooled data points. No calls are highlighted by open circles and miscalls are highlighted by filled circles. Shown on axes are arbitrary fluorescence units. B: Effect of pooling WGA products from a 300 colon cell cap on a STR-genotyping assay using a 2-bp repeat marker D13S173.
Figure 5
Figure 5
Relative gene copy number analysis using an unamplified genomic DNA as a standard. Pooled WGA products from microdissected colon cells were analyzed using TaqMan quantitative PCR assays and compared with a WGA product from 5 ng of genomic DNA isolated from cultured cells. The copy numbers are averages of four or six amplified samples, and the error bars show corresponding SD.
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