Award details

Improving the specificity and throughput of automated analysis of chromatin fine structure in eukaryotic cells

Reference	BB/E005640/1
Principal Investigator / Supervisor	Professor Constanze Bonifer
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	School of Medicine
Funding type	Research
Value (£)	65,244
Status	Completed
Type	Research Grant
Start date	13/11/2006
End date	12/08/2008
Duration	21 months

Abstract

The binding of regulatory proteins can be studied by exposing the cells to agents that chemically modify the DNA which is then cleaved at the point of modification. The accessibility of different types of modifying agents to DNA in living cells is affected by the presence of bound proteins, such as transcription factors and chromatin components. These 'footprints' can be visualised by Ligation Mediated/PCR (LM-PCR). The presence or absence of proteins and the alteration of chromatin patterns can be deduced by comparing the intensity of bands between the test sample and control samples. This method, though very powerful, is dependent upon the accurate binding of gene specific primers, particularly the first primer. Any non-specific annealing leads to high background and poor results. We propose to improve this technique by making use of Pyrophosphorilysis-Activated Polymerisation (PAP), which uses the ability of DNA polymerase to reverse the polymerisation, in the presence of high pyrophosphate. In this procedure a blocking nucleotide can only be removed if the primer is bound specifically to the DNA. This will significantly increase the specificity of the reaction and allow the use of generic labelling primers to visualize the final product, thereby reducing both the cost and the time needed for the procedure. In addition, we propose to adapt this procedure for automation. With the increasing emphasis on genome wide analysis, it is no longer sufficient to be able to investigate one gene at a time. We have previously adapted LM-PCR for use on an automated workstation thereby greatly increasing the throughput of the method. We intend to do the same for LM-PCR-PAP. In short LM-PCR-PAP would be an extremely powerful tool for analysing the binding of proteins to DNA in vivo, that would be quicker and cheaper than the current methods, and with automation be capable of analysing a large number of samples in a relatively short time frame.

Summary

The molecular mechanism of gene expression is not fully understood, but the rules are the same for all organisms. One important discovery regards the role of chromatin. Chromatin is the name for the proteins compacting the one-meter long DNA molecule so that it fits into the cell nucleus. Active genes possess areas of reduced compaction. There are protein complexes pushing chromatin aside or modify it, so that genes can 'go to work'. The details of these processes are complicated and there are still many open questions. Therefore the chromatin structure of many genes needs to be studied so we can reach conclusions about general principles of the role of chromatin in gene expression, and how chromatin responds to signals coming from outside the cells. However, genes are very large and comprise many thousand and sometimes millions of base pairs and our current methods are too slow. We therefore urgently need methods that speed up the analysis of genes. We have developed a method that allows the rapid examination of the chromatin fine structure of entire gene loci, using a robot. However, although this has greatly improved our capacity to analyze genes, there is still a lot of room for improvement. This proposal aims at doing just that: we propose to further simplify existing technology and to make our assays more specific.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	Tools and Resources Development Fund (TRDF) [2006-2015]
Funding Scheme	X – not Funded via a specific Funding Scheme

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