LEC EXAM #3 CHP. 12

Question 1

Restriction enzymes:

Answer 1

Enzymes that cuts DNA at specific sites

Question 2

Recombinant DNA

Answer 2

Takes a human gene and recombines it with bacterial DNA using a restriction enzyme

Question 3

Vector:

Answer 3

Plasmids that have a MCS (multiple cloning site) that has restriction enzymes that cut enzymes to allow insertion of gene of interest

Question 4

Genomic libraries:

Goal?

Answer 4

-46 chromosomes-> treat with restriction enzyme-> cuts into fragments-> put inside plasmid-> gets inserted into bacteria cell->
GOAL: make bacteria make multiple copies of fragments that are used for DNA sequencing

Question 5

cDNA libraries:

Answer 5

Isolates RNA> cuts it up-> use reverse transcriptase to converts RNA to cDNA-> cDNA gets inserted into plasmids-> bacteria transformed with vectors-> make multiple copies

Question 6

Electroporation:

Answer 6

Current/charge in cells that causes disruption/holes in the phospholipid bilayer that helps you get plasmids into bacterial cells

Question 7

Why is cDNA more specific than genomic libraries?

Answer 7

You know exactly what genes are being expressed because you make mRNA to make proteins

Question 8

Microinjection:

Answer 8

How we inject into human cells

Question 9

DNA probes:

Answer 9

Molecular structure that can be attached to a piece of DNA that makes it visible
(Ex: Fluorescent dye or radioactive)

Question 10

Purpose of DNA probes: (2)

Answer 10

• Used to distinguish DNA by adding a probe

- Visualization of specific sequence of DNA

Question 11

Gel electrophoresis structure:

Answer 11

• Gel agarose which is long strands that when cooled layer on top of each other
• Place well comb into agarose to form holes/mesh structure
• Put dye in DNA and load them into wells
• Buffer/current applied to the outside-> negative charge on one end and positive charge on the other end

Question 12

Goal of gel electrophoresis:

Answer 12

Separates DNA by size

Question 13

Result of gel electrophoresis:

Answer 13

• Smaller pieces of DNA move through the mesh faster to the positive charge
• Larger pieces of DNA take longer

Question 14

RFLP mapping abbreviation:

Answer 14

Restriction fragment length polymorphism

Question 15

RFLP mapping:

Answer 15

Looks at DNA that’s been cut up/digested by restriction enzymes and separates into size by gel electrophoresis

Question 16

Southern blot:

Answer 16

-DNA is separated by size via gel electrophoresis then tested to see if truly similar in DNA sequence and fragment size
-Transferred to nylon membrane
-Complimentary probe is added to DNA sequence of interest
-Dark spot on the x-ray film allows you to visualize the piece of DNA and see where its located
(probe is radioactive)

Question 17

DNA microarray process:

Answer 17

Used to isolate mRNA of human cells-> reverse transcriptase-> makes complementary strand cDNA-> red or green fluorescent probes added to complimentary DNA-> combine targets-> hybridize to microarray tray with complimentary pieces of DNA sticking up

Question 18

Purpose of DNA microarray process:

Answer 18

Used to compare human cells to cancerous cells to see what cells are being expressed

Question 19

Amount of hybridization=

Answer 19

amount of gene expression

Question 20

SDS-page

Answer 20

Similar to gel electrophoresis but separates proteins by size instead of DNA

Question 21

How to treat SDS-page?

Answer 21

With detergent that has a negative charge

Question 22

What is the gel/linear fiber made of in SDS-page?

Answer 22

Acrylamide

Question 23

PCR steps:

Answer 23

1. Heat up strand-> opens DNA
2. DNA primers anneal and bind to complimentary piece of DNA
3. Lower temp down so TAQ DNA polymerase can function
4. DNA polymerase fills in DNA

Question 24

At 72C:

PCR

Answer 24

Allows primers to bind and fills in DNA

Question 25

TAQ polymerase:

Answer 25

• made from DNA
• Comes from archae that live in thermal vents (thermophilus aquadicus)
• Able to heat up and bring back down in temp without denaturing it

Question 26

What does TAQ polymerase allow you to do?

Answer 26

Repeat making DNA

Question 27

Di-deoxynucleotides:

Answer 27

• Used for DNA sequencing to generate DNA fragments of of different lengths
• Stop replication sequencing where it picks up a di-deoxy

Question 28

Can you add onto a dideoxynucleotide?

Answer 28

No, because it’s not a free 3 prime hydroxyl

Question 29

Every di-deoxy:

Answer 29

• Has a fluorescent label/probe attached to it that comes from DNA primers
• Does not have OH group

Question 30

GFP:

Answer 30

• Green fluorescent protein produced by jellyfish

- Allows you to track location of protein

Question 31

mRNA bound by:

Answer 31

miRNA (micro RNA)

siRNA: (small interfering DNA)

Question 32

miRNA and siRNA BOTH:

Answer 32

• Made by human cells

- Regulate/stop gene expression

Question 33

RISC complex in an imperfect match:

Answer 33

Sits on mRNA and miRNA segment and blocks translation from occurring (temporary)

Question 34

RISC complex in a perfect match:

Answer 34

Stops dsRNA-> cleavages mRNA (permanent)

Question 35

Gene therapy:

Answer 35

• Genetic mutation that was inherited

- Goal is to bring in a good non-mutated copy of gene and find a virus that infects the target tissue

Question 36

Process of gene therapy:

Answer 36

Takes viral DNA and cuts it with restriction enzymes-> insert new unmodified gene ->insert into known virus-> virus infects target tissue of patient-> inserts DNA into host cell

Question 37

Plasmid contains:

Answer 37

Multiple cloning sites where you can insert your gene of interest

Question 38

Why is RISC complex temporary in an imperfect match?

Answer 38

Because it doesn’t hydrogen bond-> not a double stranded RNA structure

Question 39

Ex of vector:

Answer 39

Bacteriophage or plasmid