Automation systems aid human genome project

Highly accurate robotic systems using encoder feedback are playing a crucial role in the mapping of the human genome.

Highly accurate robotic systems, using Renishaw's RG2 encoders, have been developed by engineers at The Sanger Centre, near Cambridge, UK, to automate the inspection, picking and transferring of very small individual colonies of bacteria in a DNA sequencing facility.

The Sanger Centre is part of the human genome project, an international collaboration whose primary aim is the mapping and sequencing of the 3 billion base pairs that make up the human genome.

The project formally began in 1990 and is expected to take 15 years.

The Sanger Centre, situated on The Wellcome Trust Genome Campus in Cambridgeshire, is responsible for one third of the human genome project.

It is named after Fred Sanger, whose contribution to genomics is invaluable.

He invented, among other things, the dideoxy sequencing method; the method of sequencing still most commonly used today.

DNA sequencing is the process of determining the order letters in the DNA code - A, T, C and G.

The human genome project is effectively "writing out" all the letters in the human genome in the correct order.

The next stage in the process is to understand these letters and hence to understand the functions specified by different regions of DNA code.

The method of sequencing used at The Sanger Centre entails a number of different stages and processes.

First, DNA is broken down into smaller, more manageable pieces, which are then localised onto human chromosomes, or "mapped".

These pieces are joined to a cloning vector called a BAC that allows researchers at the Sanger Centre to grow the DNA in bacteria.

This cloning process provides the primary resource for DNA sequencing.

Each of the BACs is then, in turn, broken into thousands of overlapping pieces and each is joined to a second vector and again inserted into bacterial cells.

E Coli bacteria grow on Petri dishes into small colonies discrete populations of bacteria derived from a single cell and therefore containing a single piece of DNA sequence.

It is after this subcloning process that the sequencing reaction is undertaken.

In brief, each colony produced by subcloning is picked and grown overnight in a bacterial growth medium.

The bacteria are then broken open using detergent to release the DNA vector.

Sequencing is then undertaken by imitating the processes that copy DNA in a cell.

A primer is added to one end of the DNA strand to which complimentary bases are added.

A mixture of normal and "dideoxy" bases are added to the reaction.

A dideoxy base terminates a chain, adding a fluorescent dye to the final base, allowing it to be recognised.

The reaction is repeated many times to allow different sized molecules to be produced, each one ending in a base with a fluorescent tag.

The sample of DNA is then run on a sequencing machine, which separates the different sized molecules by using an electrical field to propel the sample through a tiny capillary tube.

The smallest molecules are drawn down fastest and the larger molecules are drawn down more slowly.

The coloured tag at the end of each molecule is detected following laser light excitation, allowing the order of the bases to be read.

The individual sequences are then reassembled and it is then up to the five finishing teams at the Sanger Centre to bridge any gaps that are not covered by the original clones.

The human genome project has agreed on a "gold standard" sequence, which will contain less than one gap in a sequence of 10,000 bases.

The Sanger Centre typically produces a sequence with less than one gap in 100,000 bases.

Following the subcloning process, and prior to the sequencing reaction taking place, is a point at which automation has become a vital part of work at the Sanger Centre.

Having produced a colony through subcloning, it is necessary to pick the individual colonies and hence individual isolates of DNA - which have grown on a Petri dish.

Previously, this process was undertaken manually, but because the high throughput of the Sanger Centre required up to 150,000 colonies per day to be picked, it was necessary to find an alternative, more efficient method of completing the picking.

The problem facing The Sanger Centre's engineering group leader, Brian Munday, however was that there was not a commercial robot of the right configuration, or robust enough, to carry out the picking, or re-arraying, on a 5 day, 12-14 hour basis.

The choice lay between small bench-mounted types or the larger gantry types, customised to suit the operations.

The solution was therefore an "in-centre" project.

The engineering team set about designing its own robotic platform that would support several applications: colony picking, sequencing reaction setup, re-arraying and microarray depositories.

Design criteria included low maintenance, high accuracy, low noise level, clinically clean, quick and easy to assemble and transportable.

A gantry design was chosen, with a carriage riding on grease-packed ball rails and linear motor drives provided by Linear Drives of Basildon, Essex.

Accurate positioning, to within 10um, and a reliable position data feedback system is crucial to the success of the operation.

These levels of accuracy are essential because each colony is less than 1mm in size.

To ensure accurate positioning, the team chose the Renishaw RG2 digital linear encoder system for the re-arraying robots.

This system was chosen because of its simplicity and the ease of installation of the tape and optical heads.

"The encoders have never let us down", said Munday.

A Baldor PC controls the linear motors and the RG2 linear encoders.

The working area under each robot is approximately 600mm deep by 1000mm wide.

A CCD camera identifies suitable colonies from each dish.

The colonies are blue if they do not contain an inserted DNA fragment but white if they do.

The robot not only uses these criteria, recognising the different colours, but also avoids picking overlapping colonies that could produce a mixed DNA sequence.

Having identified a suitable colony, a 48-pin picking tool on the robot's z-axis travelling gantry head then moves across the Petri dishes.

Each pin picks up a suitable colony and transfers it to an individual well on a 96 well plate.

Each well contains a bacterial growth medium.

The robot then moves through a series of sterilising pools to clean the pins before beginning the process again.

All operations take place in a in a low-pressure "clean room" cabinet.

The whole operation to pick colonies into 96 wells takes a few minutes.

One robot can pick and place in excess of 2000 colonies an hour.

The engineering team at the Sanger Centre has built six of these robots, each fitted with the Renishaw RG2 encoders.

A second robotic project is currently underway to further automate the sequencing operation.

It integrates three re-arraying robots with a low temperature storage system that allows the DNA template samples to be stored at 4C.

A Staubli RX90 robot traverses between the refrigerator cabinet and the row of three re-arraying robots.

The refrigerator cabinet stores over 3000 384-well microtitre plates in seven, multitiered carousels.

The Staubli robot accesses the plates from the store and transfers them to the re-arraying robots.

All three re-arraying robots are equipped with Renishaw RG2 encoders.

This part of the Sanger Centre now looks more like a factory than a laboratory.

The Sanger Centre is not only working on sequencing the human genome but also works on other sequencing projects.

It is currently sequencing the genomes of model organisms such as the zebrafish and the mouse, which can be compared with the human sequence to provide a greater understanding of it.

A cancer genome project is also underway, as are various pathogen projects.

The human genome project has already had an impact on societies worldwide.

The project will lead to better diagnostics and better treatment for disease.

It will not only lead to personalised medicines but it is hoped that it will also lead to cures for diseases.

The human genome project serves to speed up all scientific and medical advances because it makes all its data available FOC, without restriction, within 24 hours of completion.

Without automation, the task would have taken many times longer.

It would not have been possible without robotics or automation.

The engineering team at the Sanger Centre have tried to produce a platform, which does not break down.

Thanks to the reliability of system components, like Renishaw's RG2 encoders, they feel that they have achieved it.

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