DNA Technology Notes

Restriction Enzymes Scientists use several techniques to manipulate DNA (cloning = copying genes, transferring genes between organisms, etc.) DNA must first be extracted and precisely cut so that it can be studied. Restriction enzymes (or molecular scissors) cut DNA at a certain nucleotide sequence called a restriction site (4-6 base pairs long). REs come from bacteria that use them as a protection against invaders (ex. Cut up the DNA of the viruses). REs restrict (or decrease) the effect of a virus on the bacterial cell. Some REs cut straight across Some REs make staggered a DNA —> “Blunt ends” cuts —> “Sticky ends” (Velcro) Palindromic Sequence Result - DNA fragments with different lengths. The next step in the research is often the separation of the DNA fragmts by Gel Electrophoresis.

Gel Electrophoresis An electrical current separates DNA fragments by size (cut by restriction enzymes). Electricity moves DNA because DNA is negatively (-) charged and likes to go to positive (+) end. * Bigger fragments move slower * Smaller fragments are faster and are closer to positive (+) end * Gel matrix with pores — large fragments slow down. * Restriction map —the pattern of DNA fragments (bands) that shows up on the gel, which shows the sizes of DNA fragments.

Learning Check 1. If the DNA molecule in your booklet was cut at the following places, which piece would be closest to the wells (where DNA was placed into gels)? 2. DNA electrophoresis is used to study evolutionary relationships of species. According to the diagram of species A, X, Y and Z, which species has the most DNA in common with species A?

Learning Check If the DNA molecule in your booklet was cut at the following places, which piece would be closest to the wells (where DNA was placed into gels)? B closest to the wells/A farthest 2. DNA electrophoresis is used to study evolutionary relationships of species. According to the diagram of species A, X, Y and Z, which species has the most DNA in common with species A? Species Z—the fewer differences in DNA the more closely related

DNA Fingerprinting Used in a person’s identification, criminal and parental identification at the molecular level. It can show relationships among family members. In parentage– tests can not absolutely PROVE parentage but can absolutely disprove. A DNA fingerprint is a type of restriction map. 99.9% of human DNA is the same in EVERY person except monozygotic (identical twins) The greatest differences in the DNA among people are found in regions of the genome that are NOT parts of genes (Noncoding regions). DNA fingerprinting focuses on these regions. These regions include stretches that repeat. Each person’s DNA differs in the numbers of copies of the repeated sequences. The difference in the number of repeats are found by separating the DNA fragments with gel electrophoresis. The pattern of DNA fragments on a gel represents the uniqueness of a person’s DNA (“fingerprint”).

Learning Check 1. The diagram below represents DNA fingerprints which are the results of gel electrophoresis done on several DNA samples found as a crime scene. Which suspect is linked to the crime scene by this DNA analysis? 2. Each fragment of DNA is marked with a letter. A child MUST get each of its fragments from at least one of it’s parents. Is the alleged father the child’s father?

Learning Check 1. The diagram below represents DNA fingerprints which are the results of gel electrophoresis done on several DNA samples found as a crime scene. Which suspect is linked to the crime scene by this DNA analysis? Suspect C 2. Each fragment of DNA is marked with a letter. A child MUST get each of its fragments from at least one of it’s parents. Is the alleged father the child’s father? Yes possibly.

Learning Check Did the following puppies come from the indicated parents?  

Learning Check Did the following puppies come from the indicated parents? Yes those parents COULD have produced this puppy.   Puppy 2 did not come from these parents

Genetic Engineering AKA genetic modification (GM) It relies on cloning of genes (segments of DNA). In some cases, scientists insert cloned genes from one organism into a different organism. This changing of an organism’s DNA to give the organism new traits is called genetic engineering. It is based on the use of recombinant DNA technology. Recombinant DNA is DNA that contains genes from more than one organism. First GMO was in 1973– bacteria. Bacteria are commonly used because they have tiny rings of DNA—PLASMIDS. Plasmids are used as vectors (vehicles) in genetic engineering Gene to be replicated is inserted into plasmid ( often antibiotics)

PCR (Polymerase Chain Reaction) A sample of DNA is too small to work with. A specific DNA sequence must be copied, or amplified, to produce a sample that is large enough to study. PCR (Polymerase Chain Reaction) is a technology that uses DNA polymerases to produce millions and even billions of copies of a specific DNA sequence in just a few hours. ***The amplification of recombinant plasmids occurs in PCR*** How are replication and PCR similar? Different? Explain. Many human genetic diseases are caused by recessive alleles of genes. How might PCR be important in the diagnosis of these illnesses?

PCR (Polymerase Chain Reaction) How are replication and PCR similar? Different? Explain. They both copy DNA. PCR copies a specific portion over and over. Replication copies the entire DNA strand. Many human genetic diseases are caused by recessive alleles of genes. How might PCR be important in the diagnosis of these illnesses? You can design primers that will detect these alleles and you could find out if a person has that specific disease with in hours, just by collecting a small DNA sample.

Uses of Genetic Engineering The cow = GM cow that produces milk without lactose = no allergies, milk also has more proteins and vitamins Some species have been GM to produce less fat = leaner meat GloFish—available at a Walmart near you, glow under black light. DNA from jellyfish inserted into fish—fish make proteins that glow Potential risks : Government regulation. Creation of super-bug etc. GMO plants that make protein to kill insects, insects become resistant etc.

Genetic Engineering – Cloning Producing genetically IDENTICAL organisms Occurs in nature when plants, bacteria etc. reproduce asexually (by mitosis—NO partner) assuming no mutations occur. Ex. Strawberries Ex. Bacteria (binary fission) Dolly the sheep (1996) - First mammal to be cloned from a somatic (BODY) cell (taken from the udder) Other species cloned: horses, bulls, cows, pigs, and mice.

Steps to make Dolly Body=somatic cells with desired GENES Have full set of chromosomes Unfertilized egg from black faced sheep (ewe) removed and enucleated (nucleus taken out) Surrogate Body cell with the nucleus (DNA) is donated by the udder of a white-faced sheep Nuclear Donor with desired genes The body cell with the nucleus (DNA) is placed next to the empty egg. Electric shock fuses the cells. Zygote grown in a lab until it was a blastocyst Implanted into surrogate ewe. Clone!! Identical to Donor sheep What color face did Dolly have? White Why? DNA (nucleus) was donated by the white-faced sheep. Empty Egg cell with vital components In the cytoplasm. 1/2 the number of chromosomes Shocked to FUSE IMPLANTATION White-faced CLONE

Learning Check Tissue taken from a black mouse was enucleated. The resulting nuclei were then put inside the enucleated egg from a white mouse. What color would the baby mice be? Monozygotic (identical) twins are formed from a fertilized embryo splitting into 2 separate masses during development. Is this the same as cloning? What is cloning?

Learning Check Tissue taken from a black mouse was enucleated. The resulting nuclei were then put inside the enucleated egg from a white mouse. What color would the baby mice be? The mice would NOT resemble the white mouse but would be identical to the black mouse (nuclear donor) Monozygotic (identical) twins are formed from a fertilized embryo splitting into 2 separate masses during development. Is this the same as cloning? No, when twins form they are splitting a developing organism. Cloning involves placing DNA from another organism into an empty egg What is cloning? It involves placing DNA (nucleus) from a body cell of another organism, the nuclear donor, into an empty egg cell.

Genetic Engineering Types of Controlled BREEDING A. Selective Breeding (Artificial Breeding) When humans have selected desirable traits and mated organisms in an attempt to increase the trait in a population (controlled sexual reproduction). Most domestic animals. Ex. Dogs Ex: Corn What effect does selective breeding have on a population’s variation? It will usually decrease variation because we eliminate some variations in favor of an extreme

Genetic Engineering Types of Controlled BREEDING B. Inbreeding Mating of genetically similar pairs with similar characteristics (closely related—immediate family). Results in homozygosity, which can increase the chances for an offspring being affected by a recessive condition. At one time common in royalty. Ex. Hasburg chin Why is inbreeding prohibited in the AKC - purebred dog Registry (to some extent)? It decreases the vitality (health) of the dogs by making them more susceptible to genetic disorders

Hybridization Offspring resulting from the interbreeding of two different (but closely related) species to bring the best traits of both organisms. Creating hybrid plants is different than creating hybrid animals. Animal hybrids usually have reduced vigor– this is often attributed to a mismatch of the chromosome number and results in sterility if chromosome number is odd. Ex. Donkey x Horse Donkey= 62 chromosomes X Horse= 64 chromosomes = Mule (63) Sterile Many crops today are hybrids. Hybrid plants are usually stronger and healthier, with higher resistance, productivity and lifelongness: Rice, Chili, Tomato.