Test 1: Recombinant DNA Tech

Question 1

Recombinant DNA technology is used to

Answer 1

study role of protein

Question 2

studying role of protien includes (2)

Answer 2

1. determining enzymatic activity

2. determining effect of loss of protein/overexpression

Question 3

Recombination DNA tech is…

Answer 3

isolation and manipulation of genetic material

Question 4

manipulation now based on

Answer 4

genotype not phenotype

Question 5

For cloning, gene of interest must be identified by… (3) steps.

Answer 5

1. Purify protein of interest
2. sequence AA
3. search in database

Question 6

DNA microarray may be used to determine which genes are expressed in which specialized cells. It uses (protein/DNA/mRNA) that is tagged. It is hybridized with the gene chip, which is ______.

Answer 6

mRNA; glass containing gnes from cells

Question 7

Why is PCR necessary after gene of interest ID?

Answer 7

lots of DNA req for manip

Question 8

PCR requires… (5)

Answer 8

1. DNA template
2. Primers specific to gene of interest
3. DNA polymerase
4. Nucleotides

Question 9

Primers are…

Answer 9

ssDNA manifactured to hybridize to gene of interest via complementation

Question 10

PCR has 3 steps: which are

Answer 10

1. Denaturization to form ssDNA
2. Hybridization of Primer onto ssDNA
3. DNA Polymerase synthesize complement from primer.

Question 11

One round of PCR produces _ DNA copies. Two rounds _. Three rounds _.

Answer 11

2, 4, 8.

Question 12

Cloning Specific Genes (ID’d already after sequencing AA) has 3 stes which are:

Answer 12

1. Purify cell from Cell
2. Denature and Add specific Primer
3. PCR

Question 13

In cloning specific genes, only specific gene of interest are amplified because of

Answer 13

primer

Question 14

Cloning Specific Genes con is… fix it by… (2)

Answer 14

Mutagenesis.

1. Sequence fial product
2. Use proofreading polymerases

Question 15

Cloning Specific Genes with INTRONS: What is the main difference? steps (4)

Answer 15

mRNA used.

1. isolate/purify mRNA
2. Reverse transcriptase to make cNDA that is mRNA-DNA hybrid
3. Denature as with dsDNA + add primer
4. PCR on ssDNA

Question 16

Cloning Specific Genes with INTRONS: No purification after denaturization of DNA-mRNA hybrid required because

Answer 16

PCR work only on DNA

Question 17

Determine Enzymatic Activity of Protein requires lots of proteins for assays. This can be done by…

Answer 17

Adding plasmid to bacteria

Question 18

Adding plasmid with specific gene to bacteria to produce lots of protein requires 4 steps.

Answer 18

1. Plasmid must have selectible marker
2. Insert cloned gene between promotor and purification tag
3. transform plamid into bascteria
4. bacteria transcribes from primer to get mRNA WITH PURIFICATION TAG

Question 19

What is Selectable Marker? Why?

Answer 19

~drug resistance to make sure what bacteria has integrated plasmid

Question 20

What is Purification tag? How does it work?

Answer 20

Help purify protein after gene of interest is transcribed and translated. It often binds to beads after the bacteria has been lysated. The flow-through is unwanted products while the purified protein stay binded to the beads.

Question 21

What are 3 Purification tags? What does it bind to?

Answer 21

1. Histidine 6 - Binds to Ni2+
2. Glutathione S Transferase (GST) - To glutathine
3. Haemaglutinin (HA) - To antibodies

Question 22

Purification tags usually does not interfere with AA folding. If not, it must be…

Answer 22

removed with protease

Question 23

After getting purified protein, you can

Answer 23

Use cloned gene to delte gene from organism (knock-out) and Make transgenic Animal

Question 24

Making transgenic knock-out animal: 5 steps

Answer 24

1. Introduce mut plasmid to ES culture (br fur)
2. Select for cells with integrated genome
3. Inject mut ES into embryo (wh fur)
4. Chimera pups with mottled fur tested for mut gene, and hope for mut cells in germ line
5. Chimera x white fur bred. If mut germ cell, pups will have brown fur (dominant)

Question 25

Making Transgenic knock-out Animal: Brown fur != success because…

Answer 25

HETEROZYGOUS: Only 1 copy of mut gene replaced with mut gene.

Question 26

Breed homozygous mut rats by…

Answer 26

Mating homozygous mice ith homozygous mice for 25% chance of DD (not DW or WW). Then PCR. (mut wil have smaller gene)

Question 27

What is a Genetic Construct?

Answer 27

Piece of DNA that can be put into cell/animal to allow for experiments. Contains promotor, GFP, and Gene of Interest

Question 28

GFP tags 3 things, which are.

Answer 28

1. Gene of interest- which specialized cell expresses this?
2. Proteins - where in cell expresses this?
3. Promotor - which organ expresses tis?

Question 29

Labeling specific cell type with GFP is useful because

Answer 29

studying gene x to determine which differentiated muscles it is involved in.

Question 30

You made a KO mouse but dont see muc of a difference. Labeling specific cell type with GFP helps because…

Answer 30

more thorough analysis of muscle as group to compare with WT and mut mice

Question 31

Labeling specific cell type with GFP requires a structure of…

Answer 31

specific promotor next to GFP next to gene of interest. Made into trnasgenic animal to see if expressed in muscle cells.

Question 32

GFP with protein steps (3)

Answer 32

1. P-GFP -R (drug resistance) plasmid. PCR
2. to make gene of interest by adding gene between P and GFP. Transform, transcribe, translate
3. GFP tagged protein can be visible under microscope.

Question 33

GFP with promotor steps (3)

Answer 33

1. Promotor – code region – stop
2. Use PCR to clone a copy of promotor of gene of interest, and add it in front of GFP and R.
3. CLone promotor cut paste in front of GFP, transgenci animal, GFP expressed were gene of intrest is normally expressed.