PowerLecture: Chapter 16 Studying and Manipulating Genomes

Genetic Changes  Humans have been changing the genetics of other species for thousands of years Artificial selection of plants and animals Artificial selection of plants and animals  Natural processes also at work Mutation, crossing over Mutation, crossing over

Discovery of Restriction Enzymes  Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack  Discovered bacteria have an enzyme that chops up viral DNA

Specificity of Cuts  Restriction enzymes cut DNA at a specific base sequence  Number of cuts made in DNA will depend on number of times the “target” sequence occurs

Using Plasmids  Plasmid is small circle of bacterial DNA  Foreign DNA can be inserted into plasmid Forms recombinant plasmids Forms recombinant plasmids Plasmid is a cloning vector Plasmid is a cloning vector Can deliver DNA into another cell Can deliver DNA into another cell

Fig. 16-3a, p.244 Plasmids

Fig. 16-3b, p.244 Plasmids

Gel Electrophoresis  DNA placed at one end of gel  current is applied to gel  DNA has negative charge … moves toward + end of gel  Smaller molecules move faster than larger ones

Fig. 16-9a, p.249 Gel Electrophoresis

Fig. 16-9b, p.249 Gel Electrophoresis

Restriction enzymes  Resulting fragments have “sticky ends”

Fig. 16-2, p.244 Stepped Art GCTTAA AATTCG 3’3’ 5’5’ 3’3’ 5’5’ CTTAA AATTCG cut fragments G DNA ligase action nick GCTTAA AATTCG 3’3’ 5’5’ 3’3’ 5’5’ another DNA fragment AATTC 3’3’ 5’5’3’3’ 5’5’ G 5’5’ one DNA fragment 3’3’ 3’3’ 5’5’ G CTTAA 3’3’ 5’5’ enzyme recognition site GCTTAA AATTCG 3’3’ 5’5’

Making Recombinant DNA 5’5’ 3’3’ G C T T A A A A T T C G G C T T A AG 3’3’ 5’5’ one DNA fragmentanother DNA fragment 3’3’ 5’5’

Making Recombinant DNA nick 5’5’ 3’3’ 3’3’ 5’5’ GA A T T C C T T A AG nick GA A T T C C T T A AG DNA ligase action

Restriction enzyme cuts molecule of chromosomal DNA or cDNA recombinant plasmids containing foreign DNA host cells containing recombinant plasmids Same enzyme cuts same sequence in plasmid DNA Foreign DNA, plasmid DNA, and modification enzymes are mixed DNA or cDNA fragments with sticky ends plasmid DNA with sticky ends Stepped Art Fig. 16-4, p.245

Making cDNA (complimentary DNA) Fig. 16-5, p.245

Amplifying DNA  insert into fast-growing microorganisms  Polymerase chain reaction (PCR)

Polymerase Chain Reaction Double-stranded DNA to copy DNA heated to 90°– 94°C Primers added to base-pair with ends Mixture cooled; base-pairing of primers and ends of DNA strands DNA polymerases assemble new DNA strands Fig. 16-6, p. 256 Stepped Art

Polymerase Chain Reaction  Sequence to be copied is heated  Primers added - bind to ends of single strands  DNA polymerase uses free nucleotides to create complementary strands  Doubles number of copies

p.248 Recording the Sequence

DNA Sequencing Reaction Mixture  Copies of DNA to be sequenced  Primer  DNA polymerase  Standard nucleotides  Modified nucleotides

Nucleotides for Sequencing  Standard nucleotides (A, T, C, G)  Modified versions Labeled to fluoresce (different color for each base) Labeled to fluoresce (different color for each base) Structure causes DNA to stop synthesis when added Structure causes DNA to stop synthesis when added

Reactions Proceed  Nucleotides added to create complementary strands - each ends at tagged nucleotide  Result - millions of copies of varying length with 1 flourescent base at the end

Recording the Sequence T C C A T G G A C C T C C A T G G A C T C C A T G G A T C C A T G G T C C A T G T C C A T T C C A T C C T C T electrophoresis gel one of the many fragments of DNA migrating through the gel one of the DNA fragments passing through a laser beam after moving through the gel T C C A T G G A C C A DNA placed on gel Fragments move off gel in size order; pass through UV laser beam Color each fragment fluoresces is recorded

Fig. 16-8b, p.248 Recording the Sequence

Gene Libraries  Bacteria that contain different cloned DNA fragments Genomic library Genomic library cDNA library cDNA library

Using a Probe to Find a Gene  To find which bacteria in a library contains a specific gene  Need a probe for that gene (A radioisotope-labeled piece of DNA that base-pairs with gene of interest)

Use of a Probe Colonies on plate Cells adhere to filter Cells are lysed; DNA sticks to filter Probe is added Location where probe binds forms dark spot on film, indicates colony with gene

DNA Fingerprints  Unique array of DNA fragments  Full siblings can be distinguished from one another by this technique

Genome Sequencing  Sequence of bacterium Haemophilus influenzae determined  Automated DNA sequencing now main method  Draft sequence of entire human genome determined in this way

Fig a, p.250 Genome Sequencing

Tandem Repeats  Short DNA regions that differ substantially among people  Many sites in genome where tandem repeats occur

RFLPs  Restriction fragment length polymorphisms  DNA from areas with tandem repeats is cut with restriction enzymes  Variation in the amount of repeat creates variation in fragment size  Variation is detected by gel electrophoresis

Genomics  Structural genomics: sequencing of genomes of individuals  Comparative genomics: investigating evolutionary relationships

DNA Chips  Microarrays of thousands of gene sequences representing a large subset of an entire genome (p251)  Stamped onto a glass plate the size of a small business card (p251)

Fig , p.251 DNA Chips

Genetic Engineering  Genes are isolated, modified, and inserted into an organism  Made possible by recombinant technology Cut DNA up and recombine pieces Cut DNA up and recombine pieces Amplify modified pieces Amplify modified pieces

Engineered Proteins  Bacteria used to grow valuable proteins Insulin, interferon, blood-clotting factors, Vaccines Insulin, interferon, blood-clotting factors, Vaccines

Cleaning Up the Environment  Microorganisms naturally break down organic wastes and cycle materials  Some can be engineered to improve efficiency

Can Genetically Engineered Bacteria “Escape”?  Designed so that they cannot survive outside lab  Some genes will be turned on in outside environment, triggering death

p.252

Engineered Plants  Cotton plants that display resistance to herbicide  Aspen plants that produce less lignin and more cellulose  Tobacco plants that produce human proteins  Mustard plant cells that produce biodegradable plastic

Fig a, p.253 Engineered Plants

Fig b, p.253 Engineered Plants

The Ti plasmid  Researchers replace tumor- causing genes with beneficial genes  Plasmid transfers these genes to cultured plant cells foreign gene in plasmid plant cell

Fig , p.253 a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue). b The bacterium infects a plant and transfers the Ti plasmid into it. c The plant cell divides. d Transgenic plants. e Young plants with a fluorescent gene product. The Ti plasmid

First Engineered Mammals  Experimenters used mice with hormone deficiency that leads to dwarfism  Fertilized mouse eggs were injected with gene for rat growth hormone  Gene was integrated into mouse DNA  Engineered mice were 1-1/2 times larger than unmodified littermates

Transgenic Mice Fig , p.254

Designer Cattle  Genetically identical cattle embryos can be grown in culture  Embryos can be genetically modified create resistance to mad cow disease create resistance to mad cow disease engineer cattle to produce human serum albumin for medical use engineer cattle to produce human serum albumin for medical use

Fig a, p.254 Genetically Modified Animals Featherless (created by traditional cross breeding)

Fig b, p.254 Genetically Modified Animals Mira a transgenic goat that makes a human anticlotting factor

Fig c, p.254 Genetically Modified Animals

Xenotransplantation  Researchers knockout the Ggta1genes in transgenic piglets  Ggta1 gene produces proteins that human antibodies recognize  Pig’s organs are less prone to rejection by a human

Safety Concerns  Superpathogens  DNA from pathogenic or toxic organisms sometimes used in recombination experiments

Safety  Hok genes  NIH guidelines for DNA research

The Human Genome Initiative Goal - Map the entire human genome  Initially thought by many to be a waste of resources  Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes  Sequencing was completed ahead of schedule in early 2001

Using Human Genes  Even with gene in hand it is difficult to manipulate it to advantage  Viruses usually used to insert genes into cultured human cells but procedure has problems  Very difficult to get modified genes to work where they should

Ethical Issues  Who decides what should be “corrected” through genetic engineering?  Should animals be modified to provide organs for human transplants?  Should humans be cloned?