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DNA Technologies and Genomics Chapter 18 Why It Matters Snowball: Key to a Murder Biotechnology Biotechnology • Any technique applied to biological systems to manipulate processes DNA technologies isolate purify, analyze and manipulate DNA sequences • DNA fingerprinting used in forensics Genetic engineering uses DNA technologies to alter genes for practical purposes 18.1 DNA Cloning Bacterial enzymes (restriction endonucleases) form the basis of DNA cloning Bacterial plasmids illustrate the use of restriction enzymes in cloning DNA libraries contain collections of cloned DNA fragments Polymerase chain reaction (PCR) amplifies DNA in vitro Recombinant DNA DNA cloning provides many copies of a gene • Used for research or manipulation Recombinant DNA contains DNA from multiple sources joined together • Recombinant plasmids containing gene of interest can be cloned in E. coli Cloning DNA Fragments Endonucleases Restriction enzymes (endunucleases) cut DNA at specific sequences in restriction sites • Restriction fragments result • Sticky ends have unpaired bases at cuts which will hydrogen bond • Ligase stitches together paired sticky ends Restriction Enzyme EcoRI Plasmid Cloning Vectors Engineered to contain gene of interest and sorting genes • Sorting genes identify E. coli with cloned plasmid • E. coli with appropriate plasmid are ampicillin resistant and blue-white screened on X-gal Plasmid Cloning DNA Hybridization Uses nucleic acid probe to identify gene of interest in set of clones • Probe has tag for detection • Identified colony produces large quantities of cloned gene DNA Hybridization DNA Libraries Genomic libary • Clones containing every sequence in a genome • Used to isolate genes or DNA sequences Complementary DNA (cDNA) library • • • • DNA sequences made from expressed RNA mRNA extracted from cell Reverse transcriptase makes cDNA Removes introns for genetic engineering Polymerase Chain Reaction Polymerase chain reaction (PCR) • Produces many sequence copies without host cloning • Amplifies known DNA sequences for analysis • Only copies sequence of interest • Primers bracket sequence Agarose gel electrophoresis • Separates fragments by size and charge • Gel molecular sieve Polymerase Chain Reaction Agarose Gel Electrophoresis 18.2 Application of DNA Technologies DNA technologies are used in molecular testing for many human genetic diseases DNA fingerprinting used to identify human individuals and individuals of other species Genetic engineering uses DNA technologies to alter the genes of a cell or organism DNA technologies and genetic engineering are a subject of public concern RFLPs Restriction fragment length polymorphisms • DNA sequence length changes due to varying restriction sites from same region of genome • Sickle cell anemia has RFLPs Southern blot analysis uses electrophoresis, blot transfer, and labeled probes to identify RFLPs • Alternative is PCR and electrophoresis Sickle-Cell RFLPs Southern Blot Analysis DNA Fingerprinting Distinguishes between individuals • Uses PCR at multiple loci within genome • Each locus heterozygous or homzygous for short tandem repeats (STR) PCR amplifies DNA from STR • Number of gel electrophoresis bands shows amplified STR alleles • 13 loci commonly used in human DNA fingerprinting Forensics and Ancestry Forensics compares DNA fingerprint from sample to suspect or victim • Usually reported as probability DNA came from random individual Common alleles between children and parents used in paternity tests • Same principle used to determine evolutionary relationships between species DNA Fingerprint Genetic Engineering Transgenic organisms • Modified to contain genes from external source Expression vector has promoter in plasmid for production of transgenic proteins in E. coli • Example: Insulin • Protocols to reduce risk of escape Animal Genetic Engineering Transgenic animals used in research, correcting genetic disorders, and protein production Germ-line cell transgenes can be passed to offspring (somatic can not) • Embryonic germ-line cells cultured in quantity, made into sperm or eggs • Stem cells Transgenic Mice Genetically Engineered Mouse Gene Therapy Attempts to correct genetic disorders • Germ-line gene therapy can’t be used on humans • Somatic gene therapy used in humans Mixed results in humans • Successes for ADA and sickle-cell • Deaths from immune response and leukemia-like conditions Animal Genetic Engineering “Pharm” animals produce proteins for humans • Usually produced in milk for harmless extraction Cloned mammals produced by implantation of diploid cell fused with denucleated egg cell • Low cloning success rate • Increased health defects in clones • Gene expression regulation abnormal Cloned Sheep “Dolly” Plant Genetic Engineering Has been highly successful • Increased resistance to environmental effects and pathogens • Plant “pharms” and increased nutrition • Callus formation Ti (tumor inducing) plasmid from crown gall disease used as vector • Transforming DNA (T DNA) genes expressed Crown Gall Tumor Ti Plasmid and Transgenic Plants Rhizobium radiobacter disarmed so cannot induce tumors Plant cell (not to scale) Nucleus T DNA with gene of interest integrated into plant cell chromosome Regenerated transgenic plant Fig. 18-15b, p. 389 GMO Concerns Genetically modifed organisms (GMOs) are transgenic and raise certain concerns • Effect on environment • Interbreeding with or harming natural species Cartagena Protocol on Biosafety provides rules on GMOs • Stringent laboratory standards for transgenic organisms • No bacterial “escapes” from labs GMO Tobacco GMO Rice 18.3 Genome Analysis DNA sequencing techniques are based on DNA replication Structural genomics determines the complete DNA sequence of genomes Functional genomics focuses on the functions of genes and other parts of the genome 18.3 (cont.) Studying the array of expressed proteins is the next level of genomic analysis Systems biology is the study of the interactions between all the components of an organism Genome Analysis Genomics • Analyzes organization of complete genome and gene networks Human Genome Project took 13 years (2003) • Revolutionizing biology and evolutionary understanding DNA Sequencing Used for small DNA sequences to genomes Dideoxy (Sanger) method of sequencing • Dideoxyribonucleotides have –H bound to 3’ C instead of –OH • DNA polymerases place dideoxyribonucleotides in DNA, stops replication • Polyacrylamide gel separates strands varying by one nucleotide Dideoxy (Sanger) Method Genomic Analyses (1) Structural genomics • Sequence genomes to locate genes and funtional sequenes Functional genomics • Studies functions of genes and other parts of genome Genomic Analyses (2) Whole-genome shotgun method • Breaks genome into many DNA fragments • Computers assemble genome based on overlapping sequences Whole-Genome Shotgun Sequencing Functional Genomics Bioinformatics • Analysis of large data sets • Uses biology, computer science, mathematics • Identify open reading frames with start and stop codons, sophisticated algorithms for introns • Sequence similarity searches Genomics revealed many unknown genes • Many genes similar between evolutionarily distant organisms Human Genome 3.2 billion base pairs Between 20,000 and 25,000 genes About 100,000 proteins • Due to alternative splicing and protein processing Protein coding only 2% of genome • 24% introns • 50% repeat sequences of no known function Genome Analysis Data mining • Gene functions • Genome organization • Expression controls Comparative genomics (with other organisms) • Tests evolutionary hypotheses DNA Microarrays DNA microarrays (chips) • • • • About 20 nucleotide-long DNA probe sequences cDNA probes made from isolated mRNA Probes red or green from different cell states cDNA from each cell state hybridize with complementary sequences on chip Used to determine how expression changes in normal and cancer cells • Also used to detect mutations DNA Microarray Analysis Proteomics Proteome • Complete set of proteins expressed by genome • Larger than genome in eukaryotes Proteomics (study of proteome) • Protein microarrays (chips) similar to DNA microarrays • Use antibodies to bind to proteins Systems Biology Studies organisms as a whole • Investigates networks of genes, proteins, and biochemistry Combines genomics and proteomics with response to environment • Complex data analysis and computer models limiting factors