A. Cloning and Sequencing the Old-Fashioned Way: PCR

Polymerase Chain Reaction: PCR
ddddddMMain Idea: PCR is valuable for its ability to amplify, or multiply, a DNA sequence of any origin. The reaction is highly specific, easily automated, and capable of working with only minimal amounts of sample.
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The Process: The numbered steps below correspond to the locations on the diagram below. Read on to find out how PCR works!
1. Isolated DNA containing the segment to be amplified is heated to 95° C to denature its strands.
2. The strands are then cooled to 37°C in the prescense of large amounts of oligonucleotide (a short strand of nucleotides, usually less than 50) primers.
3. Thermostable DNA polymerase catalyses the 5'-3' DNA sysnthesis.
4. The process is repeated until the numbers are large enough to be easily analyzed.


Figure 4: PCR Amplification Process

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Biochemists are now able to search for protein sequences and identities using the latest information via DATABASES.

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Trademark of BLAST program for nucleotide and protein searching.

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Swiss-Prot Database Logo

B. High-Tech Database Screening

GenBank
Overview: GenBank® is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences. There are approximately 22,617,000,000 bases in 18,197,000 sequence records as of August 2002. Many journals require submission of sequence information to a database prior to publication so that an accession number may appear in the paper.

For more information check out the NCBI GenBank Overview Page:
http://www.ncbi.nlm.nih.gov/Sitemap/samplerecord.html

How it works: The GenBank database is designed to provide and encourage access within the scientific community to the most up to date and comprehensive DNA sequence information.

BLAST
Basic Local Alignment Search Tool

Overview: Sequence alignments provide a powerful way to compare novel sequences with previously characterized genes. Both functional and evolutionary information can be inferred from well-designed queries and alignments, providing a method for rapid searching of nucleotide and protein databases. Since the BLAST algorithm detects local as well as global alignments, regions of similarity embedded in otherwise unrelated proteins can be detected.
Another important aspect of BLAST to note is that it has alias.
ddddddBLASTN stands for "BLAST Nucleotide"
ddddddBLASTP stands for "BLAST Protein"
They are named so as to what they especially search for!

For more information and to do your own searching, check out the BLAST page:
http://www.ncbi.nlm.nih.gov/BLAST/

SWISS-PROT Database

Overview: The Swiss-Prot Protein Knowledgebase is a product of the European Bioinformatics Institute. It is a curated protein sequence database that provides a high level of annotation (such as the description of protein function, domains structure, post-translational modifications, and amino acid sequence), a minimal level of redundancy and high level of integration with other databases.
dfdfdff When the term SwissAll is used, it is referring to a combined database that includes sequences of SWISS-PROT, TREMBL and the updated TREMBL databases. It is a smaller group than the entire SWISS-PROT database because entries here must meet three sets of criteria, as outlined above.

For more information, check out the SWISS-PROT website:
http://www.ebi.ac.uk/swissprot/index.html

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The modeling programs are able to take information from all over the world and present a family tree!

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Tree-Puzzle Logo

C. Synthetic Evolutionary Models
ddddThe programs and sources outlined above aid in the isolation and identification of genes and proteins. However, to understand trypansomes' evolutionary history, additional tools are required in order to use the genetic information gained to create family trees.

Clustal W

Overview: Clustal W is a general purpose multiple sequence alignment program for DNA or proteins.It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.
fffffffClastal W allows any input of sequences and provides the output in the form of a family tree graph. It is also part of the European Bioinformatics Institute.

For more information and to make your own family trees:
http://www.ebi.ac.uk/clustalw/

Tree-Puzzle

Overview: TREE-PUZZLE is a computer program to reconstruct phylogenetic trees from molecular sequence data by maximum likelihood. It implements a fast tree search algorithm, quartet puzzling, that allows analysis of large data sets and automatically assigns estimations of support to each internal branch. TREE-PUZZLE also computes pairwise maximum likelihood distances as well as branch lengths for user specified trees.
ddddIn addition, TREE-PUZZLE offers a novel method, likelihood mapping, to investigate the support of a hypothesized internal branch without computing an overall tree and to visualize the phylogenetic content of a sequence alignment.

For more information about TREE-PUZZLE and its newest versions:
http://www.tree-puzzle.de/