AP Biology Chapter 20DNA Technology

DNA Technology • Recombinant DNA: genes from 2 different

sources, can be 2 dif. species• Genetic engineering: direct manipulation of

genes for practical purposes• Biotechnology: manipulation of organisms or

their components for practical tasks (ancient idea)

• Gene cloning vs. cell cloning and cloning of whole organisms – including mammals– Lots of copies of a gene to be used in research– Cloning of bacteria, mice, pigs, etc for their products– Making new organisms…..?

Bacterial Tools

Restriction Enzymes• 1960’s• Cut foreign DNA• Recognize specific

sequences• Leave sticky ends• Cuts are reproducible

because they cut at specific sequences

• Fusions occur with DNA ligase

• Plasmids• Multiple copies

– “clones” of the gene

• Genes that are frequently expressed

• Can be transferred to other cells

• DNA is universal, so bacteria will produce plasmids even if DNA is not theirs originally

• Used as a “vector”

Problems and Solutions• Expression vectors are used to get prok promoter “ON”

– Make cure inserted gene is ‘downstream’ from this type of promoter

• Must remove introns for prok to read mRNA – Start with mRNA from cells that have already created the edited

version and make something called cDNA (complementary)• Use cells that aren’t bacteria/prok

– Yeast and single celled fungi and plants– Also helps with modification that occurs to proteins after

translation in euk cells• Use artificial chromosomes

– Origin, centromere and telomeres with foreign DNA• Force uptake

– Euk cells exposed to pulses of current can allow DNA through plasma membrane….

• Inject DNA– Using “shot gun approach”

Genomic Libraries

• Genomic library is a collection of all the recombinant plasmids in a particular colony of bacteria

• cDNA libraries contain all the gene that there are cDNA for

Polymerase chain reactions, etc.

• Gel electrophoresis employs electricity to separate strands of DNA based on their size

• Bands of DNA can be identified using Southern blotting – a small sequence is radioactively tagged, then put with DNA and every where it pairs up with complementary bases it can be read

• Restriction fragment length polymorphisms (RFLP’s)– Differences in Southern blot bands between homologous

chromosomes, especially in noncoding areas of genome• PCR is a technology that amplifies the DNA so there are

billions of copies– DNA from woolly mammoth– DNA from embryonic cells– DNA from crime scenes for DNA fingerprinting

Diagrams

• Be sure to read information on the following:– Gel electrophoresis pg. 374– RFLP’s and Southern Blotting pg. 375– Sanger method of sequencing pg. 378– Your lab manual – Lab 6 A and 6B

Human Genome Project

• Computer systems worked internationally for almost 15 years to list all the human genes, including the noncoding regions.

• 3 billion base pairs long• 100,000 genes• Also mapped for research and comparison

– E. coli (and other bacteria)– Saccharomyces cerevisiae (yeast)– Caenorhabditis elegans (nematode/worm)– Drosophilia melanogaster (fruit fly)– Mus musculus (mouse)– Danio rerio (zebra fish)

Terms• Genetic mapping: linkage map determined by recombination

frequencies– Currently have 500 markers on human genome

• Physical mapping: map units• DNA sequencing: list of bases for all 3million nucleotides pairs • Genome analysis

– find genes by looking for sequences, that when transcribed have a start and stop codon

– Compare sequences found in one organism and look for similar sequence in other organsims

• Microarray assay: microscope slide with known genes in wells – mRNA from a cell is obtained, reacted with cDNA, if bases match they will pair up and when hybrid DNA is placed on slide it will glow in the well if its for the gene in that well

• Genomics: the study of genomes and genes

Human Health Applications

1. Diagnosis of disease by showing a marker for the gene

2. Human Gene Therapy when there is a known allele – Enhance immune system– Fight cancer– Stem cell products

3. Pharmaceuticals– Manufacture proteins that are required – Design vaccines that are specific and harmless

Environment, Agriculture and Forensics

• Forensics uses RFLP, southern blot and gel electrophoresis to create a DNA fingerprint

• Genetically engineered microbes can ‘feast’ on oil spills and heavy metal contaminants in water and soil

• Transgenic organisms are being developed as a source of organs and as a source of medically useful proteins

• Genetically engineered plants are created to have specific useful features, as well as being pest resistant and potentially able take up N without fertilizers

Safety and Ethics

• Currently regulated by the following US agencies– USDA, United States Department of

Agriculture– FDA, Food and Drug Administration– EPA, Environmental Protection Agency– NIH – National Institute of Health

Recombinant DNA Advisory Committee

–And potentially , voters like you….