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Bioinformatics Tools: DNA

DNA Reading

Mobile Genetic Elements:

McClintock B. 1951. Chromosome organization and genic expression. Cold Spring Harbor symposia on quantitative biology 16:13-47.

 

Initial Elucidation of DNA Structure:

Chargaff E, Lipshitz R, Green C. 1952. Composition of the desoxypentose nucleic acids of four genera of sea-urchin. The Journal of biological chemistry 195(1):155-160.

Watson JD, Crick FH. 1953. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171(4356):737-738.

Wilkins MHF, Stokes AR, Wilson HR. 1953. Molecular structure of deoxypentose nucleic acids. Nature 171(4356):738-40.

Franklin RE, Gosling RG. 1953. Molecular Configuration in Sodium Thymonucleate. Nature 171(4356):740-741.

 

High Resolution DNA Structure:

Wing R, Drew H, Takano T, Broka C, Tanaka S, Itakura K, Dickerson RE. 1980. Crystal structure analysis of a complete turn of B-DNA. Nature 287(5784):755-758.

Drew HR, Wing RM, Takano T, Broka C, Tanaka S, Itakura K, Dickerson RE. 1981. Structure of a B-DNA dodecamer: conformation and dynamics. Proceedings of the National Academy of Sciences of the United States of America 78(4):2179-2183.

 

Initial Sequencing of the Human Genome:

Lander ES, FitzHugh W, Frazier M, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J et al. . 2001. Initial sequencing and analysis of the human genome. Nature 409(6822):860-921.

Venter JC, Holt RA, Yan C, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD et al. . 2001. The sequence of the human genome. Science (New York, N.Y.) 291(5507):1304-1351.

 

Population Level DNA Sequencing:

Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA, Genomes Project C. 2010. A map of human genome variation from population-scale sequencing. Nature 467(7319):1061-1073.

Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA, Genomes Project C. 2012. An integrated map of genetic variation from 1,092 human genomes. Nature 491(7422):56-65.

 

Synthetic Genomes:

Annaluru N, Cooper EM, Cai Y, Zeller K, Agmon N, Han JS, Hadjithomas M, Tullman J, Caravelli K, Cirelli K et al. . 2014. Total synthesis of a functional designer eukaryotic chromosome. Science (New York, N.Y.) 344(6179):55-58.

Gibson DG, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi Z-Q et al. . 2010. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science 329(5987):52-56.

Next Generation DNA Sequencing:

van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C. 2014. Ten years of next-generation sequencing technology. Trends in Genetics 30(9):418-426.

[Nice source for following citations describing each of the DNA sequencing methods presented.]

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DNA Research Tools

About General DNA Analysis Tools:

The basic tools included here are widely used and freely available applications representative of many similarly available tools.  In most cases, you will be able to find equivalent or even superior tools bundled in commercially available application suites.

Restriction Site Analysis:

A wide variety of molecular biological tools have been developed to move DNA sequences in and out of specific backgrounds and so restriction endonucleases (REs) no longer hold the same central role in molecular cloning.  However, they are still used for cloning and restriction fragment length polymorphism (RFLP) diagnostic analysis.

NEBcutter v2.0

Enter a DNA sequence up to 300 KB or a text file up to 1 MB in size and use NEBcutter to indicate restriction sites for selected restriction endonucleases (REs).  DNA sequences can be provided as either linear or circular and a number of preselected groupings of REs are available.  Although not obvious from its name, NEBcutter V2.0 can also predict open reading frames of indicated minimum length.

NEBcutter Guide

PCR and Sequencing Primer Design:

In general, ssDNA primer design is a bit of an empirical science.  You can design your own primers following a few simple rules.  It's also possible to use software tools to help you design primers using thos same rules.  The use of a computer implementing an algorithm is benefitial in that you can quickly generate many potential primer pairs without the introduction of expensive human errors.

However, the proof is in the pudding and you can never really know how well your primers will work until they are used in a real reaction with a real template.  With that in mind, here are a couple primer design tools that are fairly simple to use.

Primer BLAST

This robust tool is available from the National Center for Biotechnology Information (NCBI).  The application leverages the primer design program Primer3 with the NCBI's Basic Local Sequence Alignment Tool (BLAST) to provide potential PCR (or DNA sequencing) primers directed against a provided template DNA sequence.

GenScript DNA Sequencing Primers Design Tool

Enter the target DNA sequence and the desired distance to be amplified within that region and this simple web based program will return a pair or primer sequences along with their predicted melting temperatures.

Integrated DNA Technologies "Primer Quest" Design Tool

The Primer Quest Design tool produces primer pair recommendations via either a basic or an advanced interface.  Exporting the on screen output requires a free login account, but all pertinent information can be retrieved directly from the screen.  The basic interface is very similar in function to that of the GenScript DNA Sequencing Primers Design Tool.  The advanced interface allows you to adjust theoretical reaction conditions, Tm, GC content and Primer length.  Excluded and included regions of the template can also be defined.

Plasmid Map Creation Software:

There are several really excellent commercial software packages for maintaining your personally annotated plasmid and DNA libraries.  In this arena, you generally get what you pay for.  If you are a student and will only need to work with numbers of sequences that are in the low 10's of molecules, there are several freely available applications you can use like "ApE" described below.  However, if you need to create, annotate and archive large numbers of sequences, it would be best to speak with your advisor about investing in a license or two for some quality commercial software like Invitrogen's Vector NTI.

A Plasmid Editor (ApE)

This software is produced and maintained by M. Wyane Davis of the University of Utah Biology Department.

In addition to allowing you to create annotated sequence and illustration maps, the program can also read .ABI DNA sequencing files with a 4 color chromatogram view.  This is nice for migrating DNA sequencing data into cloning plasmid backbones in silico.


References:

Vincze T, Posfai J, Roberts RJ. 2003. NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic acids research 31(13):3688-3691.

Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC bioinformatics 13(1):134-134.

DNA Sequencing File Readers:

Automated dideoxy sequencing data are typically provided as .abi or .scf formated data files.  Unlike the basic text files that are often provided along with these data files, an .abi file can't be read with a simple text editor.  Because data quality can't be determined readily from a simple text file, it is often critial to review the original chromatographic data and for this, software capable of reading the .abi or SCF file is required.  Fortunately, there are several freely available sequencing file readers.

Finch TV

The Geospiza Finch TV program is freely available to academic users although download requires that you submit a registration form which includes your name and email address.  This is probably the best of the free sequencing chromatogram viewers and I'd argue that the brief inconveniece of filling out the form is well worth it.  Versions available for Windows, Mac and Linux.

Chromas Lite

Not as fully featured as the Finch TV application and only available for Windows OS, Chromas Lite is a servicable, small footprint application that does the job.

A Plasmid Editor (ApE)

This software is produced and maintained by M. Wyane Davis of the University of Utah Biology Department.

In addition to allowing you to create annotated sequence and illustration maps, the program can also read .ABI DNA sequencing files with a 4 color chromatogram view.  This is nice for migrating DNA sequencing data into cloning plasmid backbones in silico.

 

DNA Contig Generators:

Traditional dideoxy sequencing often results in sequence reads of aproximately 2000 bp of high quality information.  To obtain the sequence of regions significantly longer than 2000 bp typically requires that multiple, overlapping sequences be "stitched" together into a single contiguous sequence.  These so called "contigs" can be assembled using freely available software.

CAP3

If you have a computer with a MAC or Linux OS, you can download the program and use it directly on your computer.  If you have a Windows based machine or would just rather use an online version of the application, you can simply provide your overlapping sequence data in FASTA format in the dialog window provided at the CAP3 link, above.


References:

Huang X, Madan A. 1999. CAP3: A DNA sequence assembly program. Genome research 9(9):868-877.

NIH Office of Biotechnology Biosafety: 2013 Recombinant and Synthetic DNA Guidelines Home Page

 

As stated by the NIH Office of Biotechnology Activities:

The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) detail safety practices and containment procedures for basic and clinical research involving recombinant or synthetic nucleic acid molecules, including the creation and use of organisms and viruses containing recombinant or synthetic nucleic acid molecules.

 

This NIH Web Portal contains links to:

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