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Genomics: The Technology behind the Human Genome Project Shu-Ping Lin, Ph.D. Institute of Biomedical Engineering E-mail: [email protected] Website: http://web.nchu.edu.tw/pweb/users/splin/ Date: 12.06.2010 Decoding the Human Genome First DNA sequence-specific restriction endonuclease was discovered following accidental finding Bacterium Haemophilus influenzae rapidly broke down foreign phage DNA. Restriction enzymes led to important advances in DNA sequencing and recombinant DNA methodology. Bacteria living in hot springs have DNA polymerases that work well at extremely high temperatures made the polymerase chain reaction (PCR) molecular genetics for largescale use. Hierarchical shotgun sequencing strategy was used for sequencing the human genome. Eric S. Lander and collaborators described human genome sequence procedure published in Nature on 2/15 2001. Recombinant DNA DNA in which genes from 2 different Def: sources are linked Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products What does “Fruit code: 4 9 8” stand for? 89222: GMO product (genetically modified organism) since 1996 Such as 99222, organic fruit Such as 4922, traditional fertilized fruit (plant by herbicide) Sequencing strategies: Hierarchical shotgun sequencing Genomic library prepared No genetic or physical maps are created Restriction enzymes are used to cut DNA, and overlapping fragments are created Clones selected at random from each library and sequenced Assembler software programs organize information into genomic sequences http://www.bio.davidson.edu/courses/GENOMICS/method/shotgun.html DNA molecule Gene 2 Gene 1 Gene 3 Nucleotide Sequencing Shotgun Cloning Nucleotide sequences of thousands of mRNA-coding genes: mRNA can be purified DNA strand 3 5 A C C A A A C C G A G T (template) and complementary DNA (cDNA) made by reverse transcriptase TRANSCRIPTION Genomic DNA is much longer than a cDNA U G G U U U G G C U C A 5 mRNA 3 fragment containing a gene, and sequencing Codon long pieces of DNA continues to be a TRANSLATION challenging task. Available sequencers Gly Phe Protein Ser Trp can only sequence DNA pieces less than Amino acid 1,000 base pairs Shotgun sequencing technique: to sequence longer pieces of DNA Cut many copies of DNA randomly to generate a set of smaller, but overlapping DNA segments, sequence these segments and develop computer program to integrate sequencing data to reconstruct sequence of original DNA with no prior knowledge of where pieces originally came from Given a set of overlapping sequences randomly sampled from a target sequence, reconstruct the order and the position of those sequences in the target. Shotgun Sequencing Technique Library of DNA-segment clones is constructed by fragmenting the target genome (BAC Library) and cloning it into large fragment cloning vectors Genomic DNA fragments are then organized into a physical map Individual clones are selected, cut, and sequenced Clone sequences are assembled to reconstruct the sequence of the genome Obtain and Purify DNA Create a Set of Overlapping DNA Fragments Clone Copies of the Selected DNA Fragments Map-Cloned DNA Segments along Chromosomes Use Shotgun Sequencing for Each Cloned DNA Segment Cutting DNA with Restriction Enzyme Each restriction enzyme cleaves a specific, sequence of bases or restriction site. These are often a symmetrical series of four to eight bases on both strands running in opposite directions. If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’. Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts. Copies of a DNA molecule will always yield the same set of restriction fragments when exposed to a specific enzyme. Restriction Enzymes Used to Make Recombinant DNA Gene cloning and genetic engineering were made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations. In nature, bacteria use restriction enzymes to cut foreign DNA, such as from phages or other bacteria. Methylation, methyl groups inserted at recognition sites block restriction enzymes from cutting bacterial DNA, a covalent modification and in vertebrates is an indicator that distinguished active genes from those that are not; turn off genes. Most restrictions enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific point in these sequences. Restriction enzymes and DNA ligase can be used to make recombinant DNA, DNA that has been spliced together from two different sources. Restriction enzymes cut covalent phosphodiester bonds of both strands, often in a staggered way creating single-stranded ends, sticky ends. These DNA fusions can be made permanent by DNA ligase which seals the strand by catalyzing the formation of phosphodiester bonds.