It is often the case that an insufficient quantity of DNA can be isolated from clinical specimens and controls for full genome analysis. The quantity of DNA is particularly limited in patient tumor specimens, most notably in early tumors with limited mass and therefore, insufficient DNA may be available to perform the multiple analyses required for full genome screening [1
]. Whole genome amplification (WGA) methods have been developed to solve the problem of insufficient quantities of DNA [5
]. Using these technologies, investigators have been successful in applying genome scanning technologies to patient cohort samples collected years ago that are not recoverable by other means [7
]. WGA is useful for amplification of DNA from stored histology slides, tissue samples, and blood stains, including amplification from single sperm [8
Two common methods used for WGA are multiple-displacement amplification (MDA) and library synthesis from fragmented genomic DNA (gDNA). MDA uses the highly processive Ø29 DNA polymerase and random exonuclease-resistant primers in an isothermal amplification reaction [11
]; this method is based on strand-displacement synthesis [12
]. A second commonly used method converts randomly fragmented gDNA into a library of DNA fragments of defined size; this library can be effectively amplified several thousand-fold using a high-fidelity DNA polymerase and can be re-amplified to achieve a final amplification of over a million fold without degradation of representation [14
]. Both of these methods produce material suitable for downstream multiplex analyses [15
An advantage of the MDA method is the generation of significant quantities of large fragments of amplified gDNA in a single step. The library-based method has the advantage that it enables the creation of whole-genome DNA libraries from degraded as well as intact DNA samples. However, care should be exercised to rule out locus and allele dropouts in the WGA product. For efficient amplification, the template should be a minimum of 2000 base pairs in length and optimally greater than 10,000. A minimum of 10 ng of DNA is required [17
]. For the study described in this paper, the MDA method was used due to ease of use and perceived integrity of the circular mitochondrial DNA. The conclusions from these studies are that whole genome amplification provides a useful tool when there are limited quantities of DNA.
Although clinical testing of genomic DNA amplified with WGA techniques demonstrated excellent concordance for the detection of point mutations [18
], WGA has not been widely accepted because of concerns about replication accuracy. Further, papers reporting the use of WGA techniques have focused on the evaluation of the technique rather than on the accuracy with patient specimens. There are also a variety of factors to note when comparing WGA studies, e.g. tissue acquisition, fixation, sectioning, etc [11
]. Although mtDNA is more abundant than nuclear DNA, the same limitations are relevant with mtDNA.
Early detection of disease associated mutations frequently requires the analysis of matched body fluids samples. Often body fluids that are used to assess the presence of cancer in remote sights contain degraded DNA or very low quantity. To validate the usefulness of body fluids to potentially replace tumor tissue as a source of mtDNA, we identified a WGA system that fulfills our requirements with respect to product yield and quality and have optimized this WGA method for the entire human mitochondrial genome. Prior studies using WGA for mtDNA reported on the control region, which only accounts for 6% of the mitochondrial genome [20
]. In our study, sequence concordance for native genomic DNA and WGA-amplified DNA was analyzed using the Affymetrix GeneChip®
resequencing microarray, versions 1 and 2. These arrays have increased sensitivity over traditional fluorescent DNA sequencing for the detection of mixed bases, or heteroplasmies [22
]. Overall, the sequence identity between native and WGA material was 99.9995%. Further, for disease associated mutations (genetic differences between tumor tissue and matched blood samples) WGA correctly identified 100% of the mutations. Heteroplasmies were accurately detected using WGA. Our results show that WGA is a reliable method for amplifying mtDNA from body fluids (sputum), blood and tumor tissue.