Overview

A new method called Multiple Displacement Amplification (MDA) has made it possible to sequence bacterial genomic DNA from a single cell. MDA is used to amplify the few femtograms of DNA in the cell for use in sequencing and for diagnostic and genotyping assays. MDA is rapidly being adopted for investigation of environmental bacterial communities, such as in oceans, where it can be used to discover novel microbes. However, there has been little effort yet to use MDA for studying bacteria living in the human body where powerful new research strategies could be developed to study the normal and pathogenic flora.

Our objective is to develop the methods needed for use of MDA in amplifying bacterial genomes from human clinical specimens. Our research is focused on the basic enzymology of the MDA reaction and the chemical pathways involved in DNA amplification to improve the performance of MDA and to perfect methods and strategies for use of amplified DNA by both the Sanger sequencing method and 454 pyrosequencing. The overall goal is to develop methods for in vitro amplification of genomic DNA from single cells and to demonstrate the research applications of this new technology to the natural human bacterial flora and to infectious disease.

We are developing methods to isolate single cells from tissues, fecal samples, body fluids and other specimen types. Cells are lysed to release DNA and MDA is performed for whole genome amplification. Preliminary studies have demonstrated high throughput isolation of Haemophilus influenzae from inner ear mucosa by flow cytometry. Other pathogens to be studied include Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa from middle ear, and throat tissues and puss from wounds and infected medical implants.

Isolation of single cells is also being carried out by micromanipulation in which a glass capillary is used under the microscope and specific bacteria are targeted by labeling with fluorescent DNA probes. Microfluidic MDA on chips is being developed for use in "point of care" diagnostic tests that could be used at any location without lab processing required. Laser capture microdissection (LCM) is being tested as an alternative method of cell isolation for use with infected tissues, particularly where bacterial biofilms or intracellular infection are suspected.

Ground breaking methods are in development for combining confocal microscopy imaging of tissues and associated bacteria with single cell isolation and genomic sequencing. Bacteria from fecal samples will be used as part of a major project at Washington University in St. Louis to identify the microbes of the human gut. Borrelia burgdorferi, the cause of Lyme disease, has also been isolated from tick gut and DNA sequencing and genotyping demonstrated. Isolation from the characteristic Lyme disease rash in human skin is planned next and ultimately from human brain tissue where it is suspected of causing neurological symptoms. In parallel with the development of these new applications, our work in developing and optimizing the MDA method continues.

Funding

NIH / National Human Genome Research Institute 

Roger Lasken

Human Microbiome Project

Isolation of FISH labeled bacterial cells by micromanipulation. Fluorescent in situ hybridization (FISH) can be used to detect bacteria from environmental samples or human clinical specimens. The FISH probe hybridizes to the 16S ribosomal RNA of targeted cells. The method of micromanipulation is used to isolate the cell in a glass capillary. Amplification of the DNA by the MDA method provides enough DNA template to carry out genomic sequencing.