Exam 4 lectures (two combined) Flashcards
What are the four characteristics of the Phylum?
1) Notochord
2) Dorsil Hollow Nerve Chord
3) Post Anal Tail
4) Pheryngeal Pouches
What are Genetic Homologies?
Similarities in the DNA
What are the three similarities of Genetic Homologies?
1) similarities in highly conserved genes
2) similarities in noncoding sections of DNA
3) Psuedogene
What is Biotechnology?
Altering natural organisms for our benefit
What was Herman the Wonder Bull?
Genetically modified cow, carries gene for human lactoferin (protein - HLF)
What are the three processes of modifying the bull?
1) Isolate the HLF gene
2) Clone the gene
3) Stick it in cow
What are the characteristics of isolating the gene?
1) donated tissue that expresses the gene
2) digest the tissue with Lipases and Protase. These break down the protein - suspended in ETOH
What is Lipase?
An enzyme that breaks down lipids.
Are nucleic acids soluble in ETOH?
No.
What do you do for the nucleic acids in ETOH?
1) Centrifuge
2) Collect pellet
3) Remove ETOH - add H2O - suspended
Describe the Poly T Column
1) Extract mRNA from solution
2) Put it through thymine and A-train. DNA, rRNA, and tRNA are trash
3) Flush with weak acid (collect out flow)
4) Add nucleotids - reverse transcription
Describe cloning the gene
1) Plasmid - small non-essential extra loops of DNA in Cytoplasm (contains genes, gets replicated with cell division)
2) Restriction Endonuclease - enzyme (indentify and destroy foreign DNA)
What does the EcoR bind to?
Binds to very specific nucleotide sequence (GAATTC) and cuts the DNA at that point
Where is the sequence split?
G - AATTC and CTTAA - G
Describe the HLF process
HLF - add EcoR1 linker + DNA Ligase –> splits sequence
What are the functions of Tag Sites?
Allows to cut while keeping promotor site
Recombinant Plasmids do not attack what?
Methylated DNA
Describe the entire process of isolating and cloning the gene.
1) Isolate mRNAs from cells in pituitary gland
2) Use reverse transcriptase to synthesize a cDNA from each mRNA
3) Attach a restriction endonuclease recognition site to ends of each DNA
4) Cut cDNAs and plasmids with restriction endonucleases; remaining sticky ends join by complementary base pairing
5) Ligate cDNAs and plasmids with DNA ligase
6) Introduce recombinant plasmids into E coli cells via treatment that makes cells permable to DNA. Grow cells in presence of antibiotic. Cells that can grow become part of cDNA library. Each cell contains one type of recombinant plasmid and thus one cDNA
What do you do if you want to clone it again?
Put it back into Bacteria to divide
What all are in the plasmid?
HLF gene, two EcoR1 sites, two Tag 1 sites, and AMP
What is an RE difficient E coli?
They do not have RE and won’t have the ability to attack the bacteria
What can you do to get the Bacteria to take up the plasmid?
1) Cold wash of CaCl2 - disrupts cell wall and membrane and punches holes
2) Electroportation - hook up culture and shock it with pulses to punch holes
What do you do for the Ecoli to take up the plasmid after punching the holes?
Pour the culture on a Ager plate containing Ampicillin.
What happens if the Ecoli doesn’t take up the plasmid? If it does?
It dies. If so, sample living colonies form
