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DNA PReDuST is the most comprehensive and efficient tool available for in-silico digestion. This is the only tool that intelligently suggests combinations of restriction enzymes (RE) based on the input parameters, viz., desired fragment length, restriction enzyme type, etc., in addition to addressing all the above mentioned limitations in existing tools It produces a list of enzyme combinations. Users can also generate maps of the fragments and sequence detail information.

One of the most challenging pain points overcome by this tool is the masking of polymorphic/ mutation sites. This is highly relevant when designing Oligonucleotide probes/ primers for specific DNA/ RNA sequence. So effectively this tool can mask any specific site and then generate a list of combinations of restriction enzymes that do not contain polymorphic sites in their restriction site.

With PReDuST, laboratory conditions are mapped to specificities of restriction enzymes to find their suitability. This reduces the number of enzymes as well as firmly ensures their compatibility. The sensitive sites provided by user are masked and enzymes cutting on these sites are further removed from analysis. The combinations of enzymes are generated for user defined fragment sizes.

The complexity of mapping restriction sites of enzymes on DNA sequence is taken care by suffix tree data structure. DNA PReDuST is build on top of Microsoft Biology Foundation (MBF). MBF implements a range of parsers for common bioinformatics file formats; a range of algorithms for manipulating DNA, RNA, and protein sequences; and a set of connectors to biological Web services such as NCBI BLAST. It provides well-defined biology object model along with suffix tree implementation, where DNA PReDuST takes advantage of using MBF.

Further, .NET 4.0 allows parallel execution environment, which is used in MBF. WPF in .NET takes care of the visualization on restriction map fragment, size distribution and sequence graphs.

System Requirements
As defined by .net 4.0 RC requirements:

Supported Operating Systems: Windows Server 2003; Windows Server 2008; Windows 7, Windows Vista; Windows XP
Processor: 400 MHz Pentium processor or equivalent (Minimum); 1GHz Pentium processor or equivalent (Recommended)
RAM:96 MB (Minimum); 256 MB (Recommended)
Hard Disk: Up to 500 MB of available space may be required
CD or DVD Drive: Not required
Display: 800 x 600, 256 colors (Minimum); 1024 x 768 high color, 32-bit (Recommended)
Software: Microsoft .NET 4.0 RC

Download PReDuST

Historical background

A restriction enzyme is an enzyme that cuts double-stranded DNA. It is also known as Restriction Endonuclease because it restricts the Bacteriophages from prospering in bacteria. The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans, Werner Arber and Hamilton Smith for the discovery of Restriction Endonucleases, leading to the development of recombinant DNA technology. Restriction enzymes are one of the most useful tools available in field of genetic engineering and molecular biology. They are very frequently used in gene cloning experiments, finding specific disease related SNPs, DNA fingerprinting, DNA sequencing, Physical Mapping of DNA, Hybridization and many other biological experiments. They are also used in varied molecular biology and genetic engineering techniques such as southern blotting, northern blotting, western blotting, eastern blotting etc.

In in silico restriction digestion, the actual in vitro experimental conditions for restriction digestion of a nucleotide sequence are simulated in a computer. Here also, like in vitro experiments, ambient conditions play a pivotal role in determining the experimental outcome.

Limitations of existing solutions

The main drawbacks of currently available solutions are that they neither account for ambient laboratory conditions nor do they find combinations of restriction enzymes to be used to get desired results. They often lack in considerations like pH tolerance and temperature compatibility between enzymes, and do not provide user with any information about the generated fragment size as a result of restriction digestion of nucleic acid sequence. It is usually the user’s responsibility to check out various combinations of restriction enzymes so as to get desired range of fragments which can be used in other experiments.

Certain sites in nucleotide sequence are of utmost importance to experimental biologists as they can unveil the mystery behind some biological trait that is missing in other organisms of same species. These important mutation sites are often not considered with due importance with currently available tools. Presence of polymorphic locations in the nucleotide sequence can lead to unexpected results in experimental conditions as restriction enzymes’ activity is sensitive to these variations in sequence.

Most currently available Restriction Digestion tools are computationally very intensive and use high computational resources to digest even a small sequence. It has a reverse exponential impact on the performance with marginal increase in sequence size.

Reference

MBF : Microsoft Biology Foundation (http://mbf.codeplex.com/)
Pingoud, A. and Jeltsch, A. (2001) Structure and function of type II restriction endonucleases. Nucleic Acids Res., 29, 3705–3727
Vincze, T., Posfai, J. and Roberts, R.J. NEBcutter: a program to cleave DNA with restriction enzymes Nucleic Acids Res. 31: 3688-3691 (2003)
Roberts,R.J., Vincze,T., Posfai,J. and Macelis,D. (2005) REBASE—restriction enzymes and methyltransferases. Nucleic Acids Res., 33, D230–D232.

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