Dideoxy chain termination method and the human genome project MichAEL mCiNNIS & Gerson Mozo Mrs. Sintich AP Biology DNA Technology Project

M & M Human Genome Solutions

What is Dideoxy chain termination? Dideoxy chain termination is a method for sequencing DNA accurately using radioactive or fluorescent indicators during DNA transcription. This is done with chain terminating dideoxynucleotides, or nucleotides that bind the DNA sequence but do not allow transcription to continue. This allows scientists to verify the exact sequence of DNA since each indicator will terminate at a certain spot and emit a very distinct signal, differentiating it from the other nucleotides in the sequence. This technology is what the human genome project used to sequence their DNA.

Dideoxy chain termination diagram

Step one: preparation of reaction mixture There reaction mixture for dideoxy chain termination is made up of primer, a DNA template, dideoxynucleotides with fluorochromes (terminate the sequence), as well as deoxynucleotides which make up the sequence of DNA being identified before it is terminated

Step Two: primer elongation and chain termination During this step, the DNA is allowed to replicate, adding to the primer until a di-deoxynucleosidetriphosphate is bound to the strand, terminating replication and causing the strand to end at that point. This occurs with all strands in the solution until there are either no strands left or all chain terminating particles have bound to the strands in the solution.

Step three: capillary gel electrophoresis separation of DNA fragments After all segments have terminated and step two is complete, the strands are placed inside a solution to undergo gel electrophoresis. This will separate the strands based on physical size, which is necessary for step four.

Step Four: laser detection of fluorochromes and computational sequence analysis In this step, the gels are run through a laser which detects the location and identity of the indicators (fluorochromes) and inputs this data into a computer for analysis. This data can also simultaneously input into a chromatograph for easier interpretation of the data

Current uses useful applications of DNA sequencing include single nucleotide polymorphism (SNP) detection (detecting single nucleotide changes in DNA between strands or members of a population), single-strand conformation polymorphism (SSCP) heteroduplex analysis (mutation detection between strands of equal length), and short tandem repeat (STR) analysis (short lengths of nucleotides repeated many times). Resolving DNA fragments according to differences in size and/or conformation is the most critical step in studying these features of the genome.

Ethical concerns There are quite a few ethical concerns regarding DNA sequencing in the public and private sector: Confidentiality of DNA composition for those whose DNA was sequenced/analyzed Not knowing if the DNA of those sequenced will be used just for research purposes or for other means (cloning, gene therapy, etc.) Whether consent must be issued if the DNA of a person is to be sequenced.

Case Study The Human Genome project was an ambitious undertaking that occurred from 1990 to 2003, and was focused upon the sequencing of the entire human genome. This project started with genome annotation using expert annotators, and eventually the technology increased to the sanger method being used(dideoxy chain termination) and eventually used RNA sequencing in 2008 after the project was declared complete., which is much faster. For most of the human genome project, however, The Sanger method was used as it is a very accurate way of sequencing the DNA