Biotechnology

Question 1

what is gene cloning?

Answer 1

Take a single gene from one organism and put it in another

Question 2

What are some limitations of selective breeding tech?

Answer 2

1) wait a long time for the right gene to mutate into new form
2) be able to cross two closely related species, each of which has some phenotypes you want

Question 3

What are two reasons to use genetic eng to move genes around?

Answer 3

1) Make organism express a new phenotype

2) understand how the gene itself works

Question 4

Explain the type 1 diabetes example:

Answer 4

• eat digest food and produce lots of glucose
• glucose enters blood stream and get absorbed by cells
• cells metabolize glucose to produce energy (some stored in liver as glycogen)
• body regulates the correct amount of glucose using beta islet cells (in pancreas)
• Beta cells sense amount of glucose and release hormone INSULIN which signals to rest of the body to absorb glucose from blood stream

Question 5

What happens when you are low of glucose?

Answer 5

• Different cell in pancreas releases diff hormone - glucagon which tells live r to release glucose into blood for the cells to absorb

Question 6

How is diabetes caused?

Answer 6

• beta islet cells are destroyed by autoimmune reaction - so the body does not know if glucose is being served

Question 7

What were some issues with the pig/insulin experiment? and what is the solution?

Answer 7

1) where do you get the human gene?
2) how to you put it into another organism so that it would be inherited?
3) how do you get the host organism to recognize it as a gene and express it?
Solution: make the gene, put it in the plasmid and then transform it into bacteria.

Question 8

When replication the genes, what are two problems that came out of it?

Answer 8

problem 1) need to make a gene without introns, so make DNA copy of mRNA where introns have already been removed. So have to use cDNA.
problem 2) Need to find insulin mRNA. it is mixed with all other mRNA’s. You do not find it, you convert all mRNA’s into cDNA and clone them all. THEN you try and find the insulin.

Question 9

How do you put cDNA’s into bacteria?

Answer 9

• first put them into plasmids and then it replicates to bacteria
• plasmids that you use are called vector

Question 10

How do you get plasmid vector to carry your genes?

Answer 10

• cut plasmid that is now linear, attach end of a cDNA to end of plasmid recreating a circle

Question 11

What is the advantage of sticky end over overhanging end?

Answer 11

• overhanging part tries to hybridize to a complementary overhanging part
• sticky ends are more efficient because sticky ends stay together

Question 12

What do you need in order to attach cDNA to plasmid vector?

Answer 12

DNA ligase

Question 13

What is a transformation maker?

Answer 13

Phenotype that allows you to indentify organism that have been transformed

Question 14

How is the cDNA library created?

Answer 14

• Spread bacteria on plates w/ ampiallin, each transformed bacterium will grow/divide which creates the colony of bacteria

Question 15

How do you find the colony that has the plasmid with the insulin gene?

Answer 15

Option 1) you know the amino acid sequence, make DNA probe.
(probe=piece of DNA you use to find complimentary DNA)
Melt DNA, complimentary regions find each other, probe is complementary to insulin gene, so they should find each other.

Question 16

How do we make bacteria express insulin gene?

Answer 16

• Give it a promoter and terminator sequence so that bacteria - RNA polymerase recognizes the gene

Question 17

How do we make the insulin cDNA stay stuck to the vector?

Answer 17

• add ligase from phosphpodiester bonds

Question 18

If the sticky ends do not stick to each other, how do we solve this problem?

Answer 18

• agarose gel electrophoresis
• idea: DNA is negatively charged, move through field toward positive charge electrode (cathode)
• this forces DNA to move through thick tangle of fibres, find DNA with right size, make protein

Question 19

What does the restriction site that the clone gene is in between consist of?

Answer 19

lac of promoter and lac operator terminator

Question 20

What is the purpose of positional cloning?

Answer 20

• the purpose is to discover biochemical basis of inherited disease (cystic fibrosis)

Question 21

What is cystic fibrosis?

Answer 21

• Mandelin trait characterized mainly by respiratory infections, result in not being able to move lungs
• common among caucasians 1/2500
• recessive
• 1/25 caucasians carry mutant allele

Question 22

What is gene mapping?

Answer 22

• find out what chromosome the gene is on

- you narrow the position of the gene down to 5% of DNA in a cell

Question 23

What are the two problems that come with mapping cystic fibrosis in humans?

Answer 23

problem 1) not many traits in humans that show simple mandelian inheritance
problem 2) cannot control matings in people

Question 24

What is the solution to problem 1 of mapping cystic fibrosis (not many traits in humans that show simple mandelian inheritance)

Answer 24

• RLPs will show changes

- introduce random restriction sites, look for the changes,

Question 25

How do you test for the exact presence of RFLP?

Answer 25

- PCR (polymerase chain reaction) - method: produce large quantities of short stretch DNA - Idea: make two DNA strands, one primer complementary to each other strands - repeat about 40x, then a lot of DNA created, turn it into gel and see it. Cut it, make it into band (s) - NOW you can tell if it is a homozygote from heterozygote (heterozygote have long and short bands)

Question 26

What is the solution to problem #2? (Can't control matings in people)

Answer 26

- look at families in which disease runs + look for linkage