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Genome Sequencing & App. of DNA Technologies • Genomics is a branch of science that focuses on the interactions of sets of genes with the environment. – Identification of Genes • computer software scans for the existence of certain known features associated with genes – start and stop sequences – intron splicing sites – ESTs - expressed sequence tags » known gene sequences expressed in functioning genes Non-Coding Areas & Mutations • Within the human genome the majority of the DNA (~75%) is made up of sequences not involved in coding for proteins, RNA, or regulatory sequences… “Junk DNA". – exon regions - 1.5% • much exists as multi-gene families • Ribosomal RNA will have hundreds of gene copies in the same gene transcript – regulatory regions - 24% DNA Fingerprinting • Segments can then be compared to find the differences by gel electrophoresis - southern blotting – Looks at non-coding sections of DNA called RFLPs (repeat fragment length polymorphisms) • RFLPs common to all of a species but differ in length within family lines – fluorescent or radioactive markers are then added to the gel to elucidate the bands present DNA Sequencing: • The human Genome Project that spanned from 1993 to 2003 pushed the development of faster more efficient methods of DNA sequencing. – Dideoxy Chain-Termination Method – DNA to be sequenced is digested and amplified (phage vectors, YACs & BACs) – Cloned fragments are then sequenced using the dideoxy method • The project continues to identify the genomes of additional organisms • Identification of Genes – computer software scans for the existence of certain known features associated with genes • start and stop sequences • intron splicing sites • ESTs - expressed sequence tags – known gene sequences expressed in functioning genes Genomic Patterns • As the complexity of the organism increases so does the amount of non-coding DNA, the amount of genes per Mb decreases • may be due to the role of non-coding regions in the regulation of gene expression • larger intron regions lead to alternative splicing and increased complexity of protein products organism haploid genome size (Mb) # of genes genes per Mb E. coli 4.6 4,400 940 nematode 97 19,000 200 drosophila 180 13,700 76 zebra fish 1,700 22,000 13 human 2,900 25,000 10 Determining Gene Function • Interactions of groups of genes – accomplished through the use of micro arrays (DNA chips) • all known genes are attached to a class slide • mRNA are harvested and copies made using cDNA, reverse transcriptase, and fluorescent labeled nucleotides • cDNA mixture is added to the slide displaying the genes active at that particular time Applications of DNA technology • Medicine – diagnosis of disease – gene therapy – development of pharmaceuticals • • • • Forensics Environmental cleanup Agriculture Applications Safety & ethical questions – GM organisms & food – privacy issues – cloning