Ch 9: Biotechnology and Recombinant DNA

Question 1

What is the function of restriction enzymes?

Answer 1

• Cut DNA at defined positions close to or within their recognition sequence
• Typically recognize 4, 6, or 8 base sequences

Question 2

True or false. Blunt ends of DNA can only ligate with specific blunt ends.

Answer 2

False. Any blunt ends can ligate together

Question 3

True or false. Stick-end DNA overhangs must be complementary in order to ligate.

Answer 3

True

Question 4

Why do bacteria produce restriction enzymes?

Answer 4

They restrict the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host cell by cleaving it

Question 5

How is host DNA modified to protect it from its own restriction enzymes?

Answer 5

Methylation of their sequences at C or A nucleotides

Question 6

What E. coli plasmid vector is commonly used for cloning?

Answer 6

pUC19

Question 7

What protein does the gene lacZ encode for?

Answer 7

β-galactosidease

Question 8

What protein does the gene ampR encode for?

Answer 8

Ampicillin resistance

Question 9

What stain is used to visualize DNA on an agarose gel?

Answer 9

Ethidium Bromide

Question 10

What are some methods of transformating DNA into cells in the lab? (5)

Answer 10

1. Electroporation
2. Chemical transformation
3. Protoplast fusion
4. Gene gun
5. Microinjection

Question 11

What is the difference between transformation and transfection?

Answer 11

Transformation: uptake of DNA into a prokaryotic cell; term means something different in mammalian cells

Transfection: uptake of DNA into mammalian cells

Question 12

True or false. Most cell types don’t naturally transform.

Answer 12

True

Question 13

How does electroporation work?

Answer 13

• Controlled short, but powerful electrical pulse induces temporary hydrophilic pores in in the cell membrane
• DNA can enter the cell
• Applicable to all cells
• Organisms with cell walls may require conversion to protoplast

Question 14

Explain how chemical transformation works in E. coli

Answer 14

• Cells incubated in ice-cold calcium chloride
• DNA is added
• Mild heat shock

Question 15

Explain how chemical transformation works for yeast

Answer 15

• Incubated in ice-cold lithium chloride
• DNA added
• Mild heat shock

Question 16

Rates of protoplast fusion increase in the presence of _____

Answer 16

Polyethyleneglycol (PEG)

Question 17

How does a gene gun (or biolistic transformation) work?

Answer 17

• Microscopic gold or tungsten particles (1 μm) coated with DNA propelled into cells with burst of helium
• If a particle lands on the nucleus the DNA may be incorporated into the chromosome

Question 18

In what organisms is biolistic transformation typically used? (3)

Answer 18

1. Plant cells
2. C. elegans
3. Yeast mitochondria

Question 19

How does microinjection for transformation work?

Answer 19

• Uses glass micropipette to inject DNA into the cell

Question 20

True or false. Microinjection is most commonly used for bacterial and fungal transformations.

Answer 20

False. Bacteria and fungi are too small. This method is impractical.

Question 21

What is a DNA library?

Answer 21

A collection of DNA fragments of one organism, each carried by a plasmid or virus and cloned in an appropriate host

Question 22

What are the two types of DNA libraries?

Answer 22

1. Genomic library: DNA fragments representing an entire organism’s genome
2. cDNA library: only complementary DNA molecules synthesized from mRNA molecules in a cell

Question 23

What nucleotide sequence does the restriction enzyme Sau3A recognize?

Answer 23

GATC

Question 24

In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 95% probability?

Answer 24

5 genome equivalents

Question 25

In genomic library construction, how many genome equivalents are necessary to ensure that all genomic sequences will be represented with 99% probability?

Answer 25

10 genome equivalents

Question 26

cDNA is made from mRNA by what enzyme?

Answer 26

Reverse transcriptase

Question 27

What are oligonucleotides (ssDNA)?

Answer 27

Synthetic DNA that can be made chemically (not enzymatically) on a solid matrix using a DNA synthesis machine

Question 28

What are some limitations of ssDNA?

Answer 28

* Limited to less than 200 bases in length * Gene sequence will be ambiguous when only the protein sequence is available

Question 29

What cell types are commonly used to make gene products?

Answer 29

1. *E. coli* 2. *Saccharomyces cerevisiae* 3. Plant cells and whole plants 4. Mammalian cells

Question 30

What are some disadvantages of using *E. coli* to make gene products?

Answer 30

1. Need to eliminate endotoxins from the products, since *E. coli* is gram-negative 2. Does not normally secrete products; need to lyse the cells to get products, which is expensive on an industrial scale

Question 31

What bacteria is alternatively used instead of *E. coli* to harvest gene products via secretion rather than lysing?

Answer 31

*Bacillus subtilis*

Question 32

What are some advantages of using *Saccharomyces cerevisiae* to make gene products? (4)

Answer 32

1. Easily grown with well-understood genomics 2. Can carry plasmids 3. Greater chance of expressing and correctly modifying eukaryotic genes and proteins 4. Likely to continuously secrete products

Question 33

What are some advantages of using plant cells to make gene products? (3)

Answer 33

1. May express eukaryotic genes easily 2. Low risk of product contamination by mammalian pathogens 3. Large scale low-cost production

Question 34

What are some advantages of using mammalian cells to make gene products? Disadvantage?

Answer 34

Adv: 1. May express eukaryotic genes easily 2. Well suited to make proteins for medical use; secreted and low risk of toxins Disadv: 1. Harder to grow

Question 35

What are some therapeutic applications of rDNA?

Answer 35

1. Human enzymes and other proteins (insulin, etc.) 2. Subunit vaccines 3. DNA vaccines 4. Gene therapy

Question 36

What is gene therapy?

Answer 36

The introduction, removal, or change in genetic material in the cells of a patient to treat an inherited or developed disease.

Question 37

Insulin's subunits are composed of _____ amino acids.

Answer 37

21 - 30 | (per subunit, insulin has 2)

Question 38

What vector is showing promise for use in gene therapy?

Answer 38

Adenovirus vectors

Question 39

\_\_\_\_\_\_\_ is a eukaryotic biological process in which RNA molecules inhibit gene expression

Answer 39

RNA intererence (RNAi) or gene silencing

Question 40

What is the difference between gene editing and gene therapy?

Answer 40

Editing: remove, disrupt, or correct faulty elements of DNA within the gene Therapy: replaces the entire gene

Question 41

What 3 components are required for gene editing?

Answer 41

1. Cas9 2. Guide RNA 3. New designed DNA

Question 42

CRISPR stands for:

Answer 42

clustered regularly interspaced short palindromic sequences

Question 43

\_\_\_\_\_% of the human genome encodes for product, while the remainder is “junk DNA”

Answer 43

2%

Question 44

What are some of the components of “junk DNA” in the human genome? (7)

Answer 44

1. miRNA genes 2. Short tendem repeats 3. Introns 4. Telomeres 5. Transposons 6. Viral remnants 7. Pseudogenes

Question 45

\_\_\_\_\_ is the science of understanding gene function through computer assisted analysis.

Answer 45

Bioinformatics

Question 46

The bacterium _______ infects specific plants at wound sites with plasmid DNA (Ti plasmid) that contains genes for opine synthesis and tumor production (crown gall)

Answer 46

*Agrobacterium tumefaciens*