Multiple strand displacement amplification of mitochondrial DNA from clinical samples.
BACKGROUND
Whole genome amplification (WGA) methods allow diagnostic laboratories to overcome the common problem of insufficient DNA in patient specimens. Further, body fluid samples useful for cancer early detection are often difficult to amplify with traditional PCR methods. In this first application of WGA on the entire human mitochondrial genome, we compared the accuracy of mitochondrial DNA (mtDNA) sequence analysis after WGA to that performed without genome amplification. We applied the method to a small group of cancer cases and controls and demonstrated that WGA is capable of increasing the yield of starting DNA material with identical genetic sequence.


METHODS
DNA was isolated from clinical samples and sent to NIST. Samples were amplified by PCR and those with no visible amplification were re-amplified using the Multiple Displacement Amplificaiton technique of whole genome amplification. All samples were analyzed by mitochip for mitochondrial DNA sequence to compare sequence concordance of the WGA samples with respect to native DNA. Real-Time PCR analysis was conducted to determine the level of WGA amplification for both nuclear and mtDNA.


RESULTS
In total, 19 samples were compared and the concordance rate between WGA and native mtDNA sequences was 99.995%. All of the cancer associated mutations in the native mtDNA were detected in the WGA amplified material and heteroplasmies in the native mtDNA were detected with high fidelity in the WGA material. In addition to the native mtDNA sequence present in the sample, 13 new heteroplasmies were detected in the WGA material.


CONCLUSIONS
Genetic screening of mtDNA amplified by WGA is applicable for the detection of cancer associated mutations. Our results show the feasibility of this method for: 1) increasing the amount of DNA available for analysis, 2) recovering the identical mtDNA sequence, 3) accurately detecting mtDNA point mutations associated with cancer.
Maragh
Samantha
S
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. samantha.maragh@nist.gov
Jakupciak
John P
JP
Wagner
Paul D
PD
Rom
William N
WN
Sidransky
David
D
Srivastava
Sudhir
S
O'Connell
Catherine D
CD
D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis
D014408:Biomarkers, Tumor; D017931:DNA Primers; D004272:DNA, Mitochondrial; D004273:DNA, Neoplasm