Chapter 13

Genetic Technology

Selective Breeding

•      For a long time, humans have selected the best plants and animals to                      breed

•      Why?

•      Examples?

•      Milk Cows

–   1947 - produced 4,9977 lbs... of milk/year

–   1997 - produced 16,9115 lbs.... of milk/year

•      Increasing the frequency of desired alleles in a population is the essence of genetic technology

Inbreeding

•      Mating between closely related individuals

•      Why?

•      Done to make sure that breeds consistently exhibit a trait and to eliminate undesired trait

–   Creates purebred lines

•      Can be bad also

–   Can bring out harmful, recessive alleles in a “family”

Hybrids

•      It can be beneficial to create hybrids

•      For example, disease-resistant plants crossed with plants that produce bigger fruit

–   Offspring get both qualities

•      Hybrids produced by crossing two purebred plants are often larger and stronger than their parents

Test Crosses

•      A test cross is a cross of an individual of unknown genotype with an individual of known genotype (usually homozygous recessive)

•      How will this work?

–   Results when heterozygous x homozygous?

–   Results when homozygous x homozygous?

•      When is this practical?

Genetic Engineering

•      Selective breeding may take a while to produce a purebred “line”

•      Genetic engineering is a faster and more reliable method for increasing the frequency of an allele in a population

•      This involves cutting - or cleaving - DNA from one organism into small fragments and inserting the fragments into a host organism of the same or a different species

•      Also called recombinant DNA technology.

–    Connecting, or recombining, fragment of DNA from different sources

Transgenic Organisms

•      Plants and animals that contain functional recombinant DNA from an organism of a different genus

–    Ex: they grow a tobacco plant that glows from a gene in a firefly

•      3 steps:

–    Isolate the foreign DNA fragment to be inserted

–    Attach the DNA fragment to the carrier

–    Transfer the DNA into the host organism

Restriction Enzymes

•      Bacterial proteins that have the ability to cut both strands of the DNA molecule at a specific nucleotide sequence

•      Some enzymes cut straight across

–   Called blunt ends

 

Restriction Enzymes

•      Many enzyme cut in palindromes

–   Ex:  a protein only cuts at AATT, it will cut the two fragments at different points - not across from each other (called sticky ends)

•   Called sticky ends because they want to bond with things due to their “open” end

•      These sticky ends are                                beneficial, because if the                                  same enzyme is used in                                     both organisms, they will                                 have identical ends and                                      will bond with each other

Vectors

•      DNA fragments don’t just attach themselves to another fragment, they need a carrier

–    A vector is the means by which DNA from another species can be carried into the host cell

•      Vectors may be biological or mechanical

•      Biological vectors include viruses and plasmids

–    A plasmid is a small ring of DNA found in a bacterial cell

•      Mechanical vectors include micropipettes and a little metal bullet coated with DNA shot with a gene gun into a cell

Insertion Into a Vector

•      If the plasmid and the DNA fragment were both cleaved with the same enzyme, they will stick together because they have “sticky ends” 

•      A second enzyme helps this process

Gene Cloning

•       Once the fragment is in the plasmid, the bacterial makes many copies of the DNA

–    Up to 500 copies per cell

•       Clones are genetically identical copies

•       Each copied recombinant DNA molecule is a clone

•       If the plasmid is placed into a plant or animal cell, the cell reproduces that DNA also and makes those proteins coded for

 

Cloning Animals

•      Dolly was the first animal cloned in 1997

•      Since then, goats, mice, cattle, pigs, etc. have been cloned

•      Take DNA out of embryonic stem cells or zygote (enucleation)

•      Insert new DNA (germ or somatic cell nuclear transfer) with another pipette or electrical current

 

Polymerase Chain Reaction

•      A way to artificially replicate DNA

•      DNA is heated and the strands separate

•      An enzyme isolated from a heat-loving bacterium is used to replicate the DNA when nucleotides are added (in a thermocycler)

–   Makes millions of copies in less than a day

•      Why could this be helpful?

 

Sequencing DNA

•      First, PCR is done to make millions of copies

•      Separate the strands of DNA

•      Place in four different tubes with four different restriction enzymes that cut at one of the four bases (A,T,C,G)

–    A fluorescent tag is also placed at each cut

•      The fragments are separated according to size by a process called gel electrophoresis

–    Produces a pattern of                                                 fluorescent bands in                                                             the gel

•      Shows the sequence of                                                                     DNA

 

Gel Electrophoresis

•      The gel is like firm gelatin

–   Molded with small wells at one end

–   Has small holes in the gel (not visible)

•      DNA has a slight negative charge

•      A current is run through the gel and an added buffer fluid

–   DNA will move towards the positive end

•      Smaller fragments fit through the holes in the gel better and move farther

Gel Electrophoresis

Gel Electrophoresis Lab

Recombinant DNA in Industry

•      E. coli has been modified to produce an indigo dye to color blue jeans

•      Recombinant DNA has been used to help production of cheese, laundry detergent, paper production, sewage treatment

–   Increase enzyme activity, stability and specificity

Recombinant DNA in Medicine

•      Production of Human Growth Hormone to treat pituitary dwarfism

•      Insulin Production by bacterial plasmids

•      Antibodies, hormones, vaccines, enzymes, and hopefully more in the future

Transgenic Animals

•      Mice reproduce quickly and have chromosomes that are similar to humans’

•      The genome is known better

•      The roundworm Caenorhabditis elegans and the fruit fly, Drosophila melanogaster are also well understood

–   Used in transgenic studies

Transgenic Animals

•      A transgenic sheep was produced that contained the corrected human gene for hemophilia

•      This human gene inserted into the sheep produces the clotting protein in the sheep’s milk

–   This protein can then be given to hemophilia patients

Recombinant DNA in Agriculture

•      Crops that stay fresh longer and are more resistant to disease

•      Plants resistant to herbicide so weeds can be killed easier

•      Higher product yields or higher in vitamins

•      Peanuts and soybeans that don’t cause allergic reactions

 

The Human Genome

•      In 1990, scientists in the U.S. organized the Human Genome Project (HGP)

–   An international effort to completely map and sequence the human genome

•      Approximately 20,000 - 25,000 genes on 46 chromosomes

•      In February, 2001, the PGP published its working draft of the 3 billion base pairs in most human cells

•      Mini-lab, page 350 (as a class)

Linkage Maps

 

Linkage Mapping

•      The problem with this in humans, is that we have relatively few offspring

•      Geneticists mark genes that have specific sequences

•      They can follow these through inheritance and hopefully see what it does

–   If a gene is marked, not passed on and that trait doesn’t show up, it may help identify the gene

Sequencing the Human Genome

•      Genome is cloned, cut into segments, and then run through gel electrophoresis

•      Arrange the fragments and get a sequence

•      Machines can do this much faster

Applications of HGP

•      Probably the biggest application so far has been the identification of genetic disorders

•      Often done prenatal

–   Take cells from                                    amniotic fluid and                                         look for deviations

Gene Therapy

•      The insertion of normal genes into human cells to correct genetic disorders

•      Have been used for SCID (severe combined immunodeficiency syndrome), cystic fibrosis, sickle-cell anemia, hemophilia and others.

•      Scientists are hopeful his will help treat cancer, heart disease, AIDS and many other things.

DNA Fingerprinting

•      Genes are separated by segments of noncoding DNA (“junk DNA”)

–   These segments produce distinct combinations of patterns unique to each individual

•      What are the uses? 

DNA Fingerprinting

•      Small DNA sample obtained

•      Clone samples with PCR

•      Cut into fragments

•      Separated by gel electrophoresis

•      Chances of two identical matches are infinitesimally small

 

Stem Cells

•      An undifferentiated cell

–   Doesn’t have a specific function yet

•      Will eventually become differentiated

–   It will get a specific function and then can only do certain thins

•      GSLC site

Other Uses of DNA Technology

•      Look at mummies to understand them

•      Looked at Abraham Lincoln’s hair

–   MAY have had Marfan Syndrome

•      Look at fossils and compare extinct species

•      They now seem unlimited.

•      Is that a good thing?