1) Selective breeding
2) Genetic engineering – humans tinker with organisms genes a. Cloning –take haploid cel and replace with a diploid nucleus (comes from organism whos traits you want to duplicate. b. Recombinant DNA – DNA from 2 or more sources. Done by Euk cells during Meiosis. Always from same molecule. 3) Biologists first started doing recombinant DNA from a prok cell and combined it with another prok cell because it was easier. 4) DNA from a euk cell and combine it with DNA from a prok cell Features of bacteria that make them useful subjects for recombinant DNA technicians: 1) Short generation time. Divide fast
2) Easy uptake of DNA from environment (transport from environment) [Fred Griffith transformation did this]. a. Many have Calcium driven DNA pumps. (especially small DNA molecules) b. Electroporation – shock DNA to open pores in plasma membrane so DNA can enter cell c. Have small DNA molecules w/ 0 plasmids 1 ori and carry accessory genes 3) Restriction endonucleases attack DNA molecule from inside. Exonucleases – chop off 1 nucleotide at a time at the ends of the DNA molecule. Not in abundant in prokaryotes Bacterial DNA gets methylated – restriction enzymes cut only unmethylated DNA Bacterial DNA gets bacteria add CH3 to Cytosine or Thymine to protect their own enzymes
Over time they have to choose to be unmethylated or methylated to cut DNA 2 types of restrictiom enzymes used to cut plasmids
Restriction enzymes that open up plasmids at a restriction site
4-8 base pairs long
Make staggered cuts
Plasmids are opened at one restriction site. Foreign Dna treated with same restriction enzymes Naïve bacteria have no plasmids.
Plasmid with wrong foreign DNA fragment
Plasmid with gene of interest
-mix with naïve bacteria get you types of bacteria
Results of recombined plasmid exposed to naïve bacteria- the 4 results: 1: naïve bacteria – will die due to no antibiotic resistance gene (ampicillin will kill it) 2: original plasmids intact lacZ gene to beta galactose to hydrolyse x gal will turn blue 3: Plasmids with wrong gene of interest
4: plasmid with gene of interest
Nucleic Acid Probe
1) ssRNA or ssDNA
3) used to find gene of interest
4) To get a nucleic acid probe to adhere to a gene of interest it should be single stranded.
Polymerase chain reaction:
USES: Take a Eukaryotic gene and put it into a prokaryotic cell and have it make a Eukaryotic polypeptide. Expose a mRNA to reverse transcriptase to get Double stranded cDNA (cDNA = DNA made from reverse transcriptase) Then expose to a restriction enzyme – some used to open plasmid. Then mix DNA and a plasmid. Add DNA ligase to transform bacteria with a recombinant DNA cloning vector. Humulin which is used for Type I diabetes, is made from E. Coli that has been genetically altered by recombinant DNA.
1) Denaturing – heating causes DNA melting by disrupting hydrogen bonds. Yields single stranded DNA molecules 2) Annealing – cools down to attach primers to single stranded DNA template. 3) Extension – temperature depends on DNA polymerase used. It is used to amplify sequences of nucleic acid.
Automated DNA sequencer:
1. Used to map out restriction site.
2. Used to determine order of DNA (A C T G) bases.
3. Used to make 1 molecule at our “model probe”
4. Want to be radioactive
Male Reproductive System Notes:
Corona and frenum are most sensitive. The shaft is the least sensitive. An acrosome contains digestive enzymes to get to the egg in the female. It is at the top of the head of the sperm. Sertoli Cells: Also called nurse cells.
1) Maintain the blood testis barrier
a. The barrier prevents sperm from entering the blood stream. If sperm enters the bloodstream the male will build antibodies to fight the sperm in the long run making the male sterile. 2) Nurse mature sperm
3) Capable of Meiosis and Mitosis.