Technical advance

Multiple strand displacement amplification of mitochondrial DNA from clinical samples

Samantha Maragh¹ , John P Jakupciak² , Paul D Wagner³ , William N Rom⁴ , David Sidransky⁵ , Sudhir Srivastava³ and Catherine D O'Connell⁶

¹Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA

²Cipher Systems, Crofton, Maryland, USA

³Cancer Biomarkers research Group, National Cancer Institute, Rockville, Maryland, USA

⁴Division of Pulmonary and Critical Care Medicine, NYU, School of Medicine, New York, USA

⁵Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, Maryland 21205, USA

⁶Tetracore, Inc., Rockville, Maryland, USA

author email corresponding author email

BMC Medical Genetics 2008, 9:7doi:10.1186/1471-2350-9-7

Published:	7 February 2008

Abstract

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.

Conclusion

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.