The Human Genome Project
Completed in 2003, The Human Genome Project (HGP) was a thirteen year project with the main goal of determining the sequence of chemical base pairs which make up DNA and to identify and map the approximately 20,000 to 25,000 genes of the human genome from both a physical and functional standpoint. Some benefits of the HGP are to predict and prevent diseases, to develop new and improved medicines, to ensure the accuracy of diagnosis, and to improve forensic science. The HGP was coordinated by the U.S. department of Energy and the National Institutes of Health. During the early years of the HGP, the Wellcome Trust (U.K.) became a major partner, additional ...view middle of the document...
The primary method used by the HGP to produce the finished version of the human genetic code is BAC-based sequencing. BAC is the acronym for "bacterial artificial chromosome." Human DNA is fragmented into pieces that are relatively large but still manageable in size (between 150,000 and 200,000 base pairs). The fragments are cloned in bacteria, which store and replicate the human DNA so that it can be prepared in quantities large enough for sequencing. If carefully chosen to minimize overlap, it takes about 20,000 different BAC clones to contain the 3 billion pairs of bases of the human genome.
In the BAC-based method, each BAC clone is "mapped" to determine where the DNA in BAC clones comes from in the human genome. Using this approach ensures that scientists know both the precise location of the DNA letters that are sequenced from each clone and their spatial relation to sequenced human DNA in other BAC clones.
For sequencing, each BAC clone is cut into still smaller fragments that are about 2,000 bases in length. These pieces are called "subclones." A sequencing reaction is carried out on these subclones. The products of the sequencing reaction are then loaded into the sequencing machine (sequencer). The sequencer generates about 500 to 800 base pairs of A, T, C and G from each sequencing reaction, so that each base is sequenced about 10 times. A computer then assembles these short sequences into contiguous stretches of sequence representing the human DNA in the BAC clone.
Another method that was used was known as the Dideoxy Sanger Method. This method uses DNA polymerase, the same enzyme used in DNA replication, to produce DNA sequence information. DNA polymerase binds to a single-stranded DNA template and adds DNA bases to the 3′ end of the complementary DNA strand it synthesizes. DNA polymerase requires an existing primer with a free 3′ end to which it adds new DNA bases in a 5′ to 3′ manner, and it moves along the template strand in a 3′ to 5′ direction.
Researchers combined the DNA template they were interested in sequencing with DNA polymerase, a single-stranded DNA primer, free deoxynucleotide bases (dATP, dCTP, dGTP, and dTTP), and a sparse mixture of fluorescently labeled dideoxynucleotide bases (ddATP, ddCTP, ddGTP, and ddTTP) that were each labeled with a different color and would terminate new DNA strand synthesis once incorporated into the end of a growing DNA strand due to the missing alcohol group on the third carbon. The mixture was first heated to denature the template DNA strand and then this is followed by a cooling step to allow the DNA primer to anneal. Following primer annealing, the polymerase synthesized a complementary DNA strand. The template would grow in length until a dideoxynucleotide base (ddNTP) was incorporated; the conditions were such that this occurred at random along the length of the newly synthesized DNA strands. In the end, the researchers were left with a mixture of newly synthesized DNA...