Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.
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.
Rook
Martha S
MS
Nuvelo Incorporated, Cambridge, Massachusetts, USA.
Delach
Scott M
SM
Deyneko
Galina
G
Worlock
Andrew
A
Wolfe
Jia Liu
JL
D005260:Female; D005838:Genotype; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D020080:Tandem Repeat Sequences / Q000235:genetics*