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 Sequence  Analysis

This page contains a collection of tools that can be used to analyze protein and DNA sequences.  Many are linked to the Biology Workbench and other free on-line bioinformatics programs and databases.  Instructions on the use of these tools and the interpretation of the output from each program are included.  There are also cross links to theoretical background information and specific assignments.

 Seq Editor   How to use a word processor as a sequence editor.

 Searches    Search for sequences (ENTREZ), structures (PDB) or articles (MEDLINE) by keywords.

 Seq. Comparison   Compare a DNA or protein sequence to sequences in a database (BLAST).

 Alignment   Align different DNA or protein sequences.

 Restriction Map   Analyze a DNA sequence for known restriction sites.

 Translation & ORF   Translate a DNA sequence into its predicted amino acid sequence.

 Reverse Comp   Obtain the reverse complement of a strand of DNA.

 Primer Design   Calculate the melting temperature and other parameters of a DNA primer.

 Splice Sites   Identify potential Intron/Exon splice sites, promoters and polyadenylation sites in a gene.

 Genomics   Examine genomic clones for sequence tagged sites to generate contigs.

 Protein Motifs   Search a DNA sequence for know protein motifs.

 Protein structure   Predict secondary structure based upon primary amino acid sequence.

 Transmembrane   Predict transmembrane domains, hydrophobicity and signal peptides from amino acid sequence.

The Biology WorkBench (http://workbench.sdsc.edu) is a revolutionary web-based tool for biologists. The WorkBench allows biologists to search many popular protein and nucleic acid sequence databases. Database searching is integrated with access a wide variety of analysis and modeling tools, all within a point and click interface that eliminates file format compatibility problems.

One current drawback of this workbench is a lack of instruction for students.  To remedy this instructions are provided using the buttons below.  If you require instructions, the most convenient approach would be to open a second Netscape window and run the workbench in one window and this site with instructions in the other.  The easiest way to do this on a PC is to right click on the link to Biology WorkBench and select Open in New Window.

You will need to establish an account and password with the workbench (be sure to write these down).  This is free and you can store files on their server.  When you enter the workbench, select the button Session Tools.

Bwbbut

You can now either start a new session or resume an existing session.  To resume an existing session, select it from the list above and select the button Resume.

Sessbut

Sequence Analysis

Each gene has a specific sequence of nucleotides, commonly called its DNA sequence. Once we have cloned a gene into a plasmid we can determine the sequence of the DNA using the dideoxynucleotide method either manually or by using automated sequencers.

 To seqeunce DNA, the DNA is first denatured, producing a single stranded template. A specific primer is then added which binds to the template. Free nucleotides (dATP, dCTP, dGTP and dTTP), dATP labeled with a fluorescent dye or a radioactive element, and DNA polymerase are added, and DNA synthesis is begun. After a few minutes the sample is split into four new tubes, and dideoxynucleotides (ddATP, ddCTP, ddGTP and ddTTP) are added. The dideoxynucleotides then are incorporated into the growing strand of a DNA molecule and stop the reaction. One then runs these reactions on a long thin acrylamide gel and separates the different length reaction products based on their size. The smaller products will move fastest, and will be at the bottom of the gel.

 One then subjects the gel to autoradiography (manual technique) or looks for fluorescent dyes (automated technique) to determine the location of each band. The trick to reading a sequencing gel is to start at the bottom of the gel and read towards the top, the smaller fragments will be closer to the start of the sequence being read.

 One of the cornerstones to the rapid advances in molecular biology and genome research is the ability to rapidly deal with very large DNA sequences using sophisticated programs and powerful computers. The increasing power of the internet has allowed greater access to these programs and databases. There are several different forms of analysis that are commonly performed on DNA sequences.

Click on either sequencing gel to the right to learn more about the steps involved in DNA sequencing.

DNAgel

Automated Sequencing

courtesy of UW-Biotech Center

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  2014 The Board of Regents of the University of Wisconsin System.

Click here to email comments to Scott Cooper regarding this site or its links.

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Manual Sequencing