Chapter 20 – DNA Technology and Genomics

Recombinant DNA Def: DNA in which genes/nucleotide sequences from 2 different sources are combined in vitro into the same DNA molecule Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: manipulation of organisms or their components to perform practical tasks or provide useful products

DNA Cloning Permits the production of multiple copies of a specific gene or DNA segment Most practical applications utilize bacteria and bacterial plasmids

Overview of gene cloning with a bacterial plasmid

Creating Recombinant DNA Restriction enzymes (endonucleases): in nature, these enzymes protect bacteria from intruding DNA; they cut up the DNA (restriction); very specific (flash movie) Restriction site: recognition sequence for a particular restriction enzyme Restriction fragments: segments of DNA cut by restriction enzymes in a reproducible way Sticky end: short extensions of restriction fragments DNA ligase: enzyme that can join the sticky ends of DNA fragments Cloning vector: DNA molecule that can carry foreign DNA into a cell and replicate there (usually bacterial plasmids

Restriction Enzymes

Cloning Cloning of human DNA using a bacterial plasmid Same technique used to create plasmid for our transformation lab http://www.dnai.org/b/index.html

Cloning a Gene

Identifying Clone Cells How identify cells? Insert ampicillin resistance gene (ampr) into plasmid, culture cells on ampicillin, if live, have incorporated plasmid Nucleic acid hybridization, uses radioactive nucleic acid probe

Genomic Libraries Can store these genes in bacteria or viruses to create a genomic library a cDNA or complimentary DNA library is made in vitro by reverse transcription of all the mRNA produced by a particular cell cDNA is used to create plasmids

Rainbow (donor) and CC (clone) Organismal Cloning Producing one or more organisms that are genetically identical to the parent that donated the single cell Important because can generate stem cells – unspecialized cells that can reproduce indefinitely and differentiate into specialized cells Stem cells have great potential for regenerating damaged tissues

Cloning Technique Technique used to create/clone Dolly, the first mammal ever clonned.

Stem Cells Difference between adult and embryonic stem cells

DNA Anaylsis and Genomics Gel Electrophoresis Restriction Fragment Length Polymorphism or RFLP analysis Southern Blot Polymerase Chain Reaction or PCR DNA Sequencing DNA microarray of gene expression

Gel Electrophoresis Used to separate nucleic acids or proteins that differ in size or electrical charge Nucleic acids carry a negative charge (why?) so they will move towards the positive end of the porous gel 1. samples loaded in gel at negative end 2. an electrical current “pulls” the fragments towards the positive end, smaller fragments move further than larger ones DNA binding dye used to visualize the fragments

Restriction Fragment Length Analysis Useful in comparing two different DNA molecules, like 2 alleles of a gene If have a small change in bp sequence, restriction enzymes will cut at different places producing different fragments Example: β-globin allele (sickle-cell)

Restriction Fragment Length Analysis (cont.) What if when comparing different genomic DNA there are too many bands for analysis? Use southern blotting Southern Blot – combines gel electrophoresis and nucleic acid hybridization (next slide) RFLP (restriction length polymorphism) – differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns, found in noncoding regions of the DNA; because these are inherited they can be used as genetic markers for making linkage maps

RFLP and Southern Blotting

PCR Polymerase Chain Reaction Method of amplifying samples of DNA in vitro Requires: dsDNA, heat-resistant DNA polymerase, RNA primers, DNA nuclotides After 20+ cycles target DNA vast outnumbers all other DNA sequences http://www.dnai.org/b/index.html

Genome Mapping Human Genome Project – completed in 2003 3 step approach Genetic (linkage) mapping – uses recombination frequencies, the ordering of genetic markers like RFLPs Physical mapping – cutting the DNA with restriction enzymes and putting them in order, make fragments overlap and use probes DNA sequencing

DNA Sequencing Dideoxy chain-termination method ssDNA incubated with tagged nucleotides DNA synthesis is stopped with tagged nucleotide Labeled strands separated through a gel and analyzed with a fluorescence detector http://www.dnai.org/b/index.html

Gene Expression Levels DNA microassay – can test thousands of genes simultaneously to determine which are expressed at any one time (tissue type, environmental conditions, stages of development, etc…)

DNA micro assay cont.

Applications of DNA Technology Medical Diagnosis of diseases Human gene therapy Pharmaceutical Products Forensic Evidence Environmental Cleanup Agricultural Animal husbandry Genetic engineering in plants