Exam 4 lectures (two combined)

Question 1

What are the four characteristics of the Phylum?

Answer 1

1) Notochord
2) Dorsil Hollow Nerve Chord
3) Post Anal Tail
4) Pheryngeal Pouches

Question 2

What are Genetic Homologies?

Answer 2

Similarities in the DNA

Question 3

What are the three similarities of Genetic Homologies?

Answer 3

1) similarities in highly conserved genes
2) similarities in noncoding sections of DNA
3) Psuedogene

Question 4

What is Biotechnology?

Answer 4

Altering natural organisms for our benefit

Question 5

What was Herman the Wonder Bull?

Answer 5

Genetically modified cow, carries gene for human lactoferin (protein - HLF)

Question 6

What are the three processes of modifying the bull?

Answer 6

1) Isolate the HLF gene
2) Clone the gene
3) Stick it in cow

Question 7

What are the characteristics of isolating the gene?

Answer 7

1) donated tissue that expresses the gene
2) digest the tissue with Lipases and Protase. These break down the protein - suspended in ETOH

Question 8

What is Lipase?

Answer 8

An enzyme that breaks down lipids.

Question 9

Are nucleic acids soluble in ETOH?

Answer 9

No.

Question 10

What do you do for the nucleic acids in ETOH?

Answer 10

1) Centrifuge
2) Collect pellet
3) Remove ETOH - add H2O - suspended

Question 11

Describe the Poly T Column

Answer 11

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

Question 12

Describe cloning the gene

Answer 12

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)

Question 13

What does the EcoR bind to?

Answer 13

Binds to very specific nucleotide sequence (GAATTC) and cuts the DNA at that point

Question 14

Where is the sequence split?

Answer 14

G - AATTC and CTTAA - G

Question 15

Describe the HLF process

Answer 15

HLF - add EcoR1 linker + DNA Ligase –> splits sequence

Question 16

What are the functions of Tag Sites?

Answer 16

Allows to cut while keeping promotor site

Question 17

Recombinant Plasmids do not attack what?

Answer 17

Methylated DNA

Question 18

Describe the entire process of isolating and cloning the gene.

Answer 18

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

Question 19

What do you do if you want to clone it again?

Answer 19

Put it back into Bacteria to divide

Question 20

What all are in the plasmid?

Answer 20

HLF gene, two EcoR1 sites, two Tag 1 sites, and AMP

Question 21

What is an RE difficient E coli?

Answer 21

They do not have RE and won’t have the ability to attack the bacteria

Question 22

What can you do to get the Bacteria to take up the plasmid?

Answer 22

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

Question 23

What do you do for the Ecoli to take up the plasmid after punching the holes?

Answer 23

Pour the culture on a Ager plate containing Ampicillin.

Question 24

What happens if the Ecoli doesn’t take up the plasmid? If it does?

Answer 24

It dies. If so, sample living colonies form

Question 25

When you get the sample living colonies, what two things can you do with it?

Answer 25

1) Create cDNA library - keeps other cDNA stored so you do not have to reisolate/clone gene again
2) Put some in the cow!

Question 26

What is the process before using the cDNA in the cow?

Answer 26

• Culture: digest with protase lipases, suspend in ETOC, centrifuge, collect pellet, suspend in H2O

Question 27

You have the Bacterial DNA and plasmids in a tube. What do you do to get the HLF gene by itself?

Answer 27

1) Add Tag 1 to get HLF gene in it
2) Separation using Gel Electrophaiesis - Ager gel

Question 28

Describe the process with Gel electrophaiesis

Answer 28

(Box with castle groves at top. Left side has the Bacterial DNA, plasmids, and HLF gene. Right side has the Ager gel). Because genes have a negative charge, the HLF gene is attracted to the bottom because there is a negative charge. The Bacterial DNA and plasmids are filtered out and digested in the gel.

Question 29

What do you do next when you isolate the HLF gene from the bacteria DNA and plasmids?

Answer 29

You put it into the cow using a micro injection. Inject the gene directly into the nucleus of one of the cow’s eggs. (Promoter site and HLF gene).