Lecture 17 - Recombinant DNA technology

Question 1

Recombinant DNA (rDNA) molecules are

Answer 1

DNA molecules formed by laboratory methods of genetic recombination

Question 2

In cloning, fragment and plasmid DNA is treated with

Answer 2

EcoRI and NotI restriction enzymes to create sticky ends

Question 3

Two DNA molecules, cleaved with a common restriction enzyme such as EcoRI, can be ligated to form

Answer 3

recombinant molecules

Question 4

If a DNA fragment doesn’t contain a site for a restriction enzyme,

Answer 4

a short, chemically synthesized DNA linker with a restriction enzyme cleavage site can be added

Question 5

A vector is a

Answer 5

DNA molecule, which is used as a vehicle to carry foreign genetic material into a cell, where it is replicated and/or expressed

Question 6

In general, there are 2 types of DNA vector

Answer 6

1. Cloning vectors
2. Expression vectors

Question 7

Cloning vectors are used to make

Answer 7

numerous copies of the inserted foreign DNA

Question 8

Expression vectors are used to

Answer 8

express large amounts of protein from the inserted foreign DNA

Question 9

Cloning vector examples (2)

Answer 9

• Plasmids
• Bacteriophage

Question 10

Plasmids are

Answer 10

naturally occurring extrachromosomal DNA

Question 11

A cloning vector contains what 3 things?

Answer 11

1. Origin of replication
2. Selection marker
3. Restriction sites

Question 12

Promotor sequences are designed to

Answer 12

drive the transcription of large amounts of a protein-coding gene

Question 13

The gene of interest in the expression vector often contains

Answer 13

fusion tags to easily purify the expressed protein

Question 14

Expression vectors often contain a polylinker region that includes

Answer 14

several restriction sites

Question 15

Nuclease cleavage at a restriction site linearizes the circular plasmid so that

Answer 15

a foreign DNA fragment can be inserted

Question 16

Recombinant plasmids are hybrid DNA molecules consisting of

Answer 16

plasmid DNA sequences plus inserted DNA elements (such hybrid molecules are called chimeric plasmids)

Question 17

The two primary uses for alkaline phosphatase (AP) in DNA manipulations:

Answer 17

1. Removing 5’ phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme
2. In subsequent ligation reactions, this prevents self-ligation of the vector

Question 18

Alkaline phosphatase removes

Answer 18

5’ phosphate groups from DNA and RNA

Question 19

APs are most active at _____ pH

Answer 19

alkaline

Question 20

Sources of alkaline phosphatase (3)

Answer 20

1. Bacterial alkaline phosphatase (BAP)
2. Calf intestinal phosphatase (CIAP)
3. Shrimp alkaline phosphatase

Question 21

Often, one desires to insert foreign DNA in a particular

Answer 21

orientation

Question 22

Transformation in bacteria is the

Answer 22

uptake and incorporation of exogenous genetic material through the cell membrane

Question 23

Transformation requires

Answer 23

competent cells

Question 24

Competent cells are in a state in which

Answer 24

DNA molecules can pass through the membrane

Question 25

Viral vectors are used to

Answer 25

get a vector into a bacterial cell

Question 26

In viral vectors, a ________ is often used

Answer 26

lambda phage

Question 27

Transfection

Answer 27

In mammalian cells, the process of uptake and incorporation of exogenous genetic material

Question 28

3 ways DNA gets through the membrane in transfection

Answer 28

1. Physical transfection
2. Chemical transfection (most common)
3. Biological transfection

Question 29

Researchers use Ti-plasmid derivatives to

Answer 29

deliver foreign genes into plant cells

Question 30

Site-directed mutagenesis is a technique for

Answer 30

studying protein structure-function relationships, gene expression, and vector modification

Question 31

In site-directed mutagenesis,

Answer 31

the sequence of a cloned DNA molecule can be altered to produce specific changes in the expressed gene product

Question 32

Oligonucleotide-directed mutagenesis is commonly used to

Answer 32

produce specific single-base changes in a cloned gene

Question 33

Oligonucleotide mutagenesis employs a

Answer 33

short primer sequence that is complementary to the gene except for one mismatched base