Ch 18: Recombinant DNA and Biotechnology

Question 1

Restriction site

Answer 1

enzymes break bonds the specific sequence

Question 2

palindromic DNA sequences

Answer 2

sequences read the same forward and backward

Question 3

sticky ends

Answer 3

DNA that is cut unevenly (staggered cuts)

Question 4

gel electrophoresis

Answer 4

a mixture of fragments in placed in a well in a semisolid gel and an electric field is applied across the gel

negatively charged NDA fragments move towards positive end

the smaller the DNA fragment, the more it moves down

Question 5

restriction enzyme

Answer 5

cuts DNA at specific site

Question 6

recombinant DNA

Answer 6

DNA made in the laboratory that is derived from at least two genetic sources

Question 7

DNA ligase

Answer 7

catalyzes the joining of DNA fragments

Question 8

Recombinant DNA technology can be used to make

Answer 8

clones aka identical copies of genes

Question 9

requirements for a vector/host

Answer 9

autonomous replication in chosen host
selectable marker for host cells
unique restriction enzyme site the vector that is not detrimental for insertion

Question 10

Plasmid

Answer 10

circular piece of DNA
replicate independently of the main chromosome

add extra genes to the host cell’s genome (metabolic enzymes, conjugation, antibiotic resistance)

Question 11

Viruses

Answer 11

replicate independently

infected cells have a different phenotype

can be altered to attenuate some detrimental genes in the virus

can be altered to carry recombinant DNA into cells

Question 12

viruses as vectors

Answer 12

bacteriophages or retroviruses

Question 13

selectable markers

Answer 13

detecting whether or not the gene of interest has been incorporated into the DNA/host

Question 14

reporter gene

Answer 14

a gene whose expression is easily observed

Question 15

lacZ gene

Answer 15

codes for an enzyme that can convert the substrate X-Gal into a bright blue product

Question 16

transformation

Answer 16

Recombinant DNA is cloned by inserting it into host cells

Question 17

transfection

Answer 17

recombinant DNA is cloned by inserting it into animal host cells

Question 18

what is commonly used as eukaryotic hosts?

Answer 18

yeasts

Question 19

Electroporation

Answer 19

a short electric shock that creates temporary pores in membranes

DNA shoots to the + end and can enter cels

Question 20

Polymerase Chain Reaction (PCR)

Answer 20

in vitro

used to make more copies of DNA seqeuces

Question 21

Steps of PCR

Answer 21

1) DNA fragments are denatured by heating g single-stranded DNA
2) Primers (one complimentary to each strand), plus dNTPs, and DNA
polymerase are added g annealing and extension g new double-stranded
DNA
3) Repeat (heating-cooling-heating….)

Question 22

initial problem with PCR was

Answer 22

its temperature requirements

key insight was the use of Taq Polymerase

Question 23

Can we mix DNAs from different species (rDNA or recDNA)

Answer 23

yes

Question 24

What DNA functions are needed to make more

(vectors)?

Answer 24

Replication, selection, insertion site

Question 25

How do you get DNA into, and then find it, in a host that can make more?

Answer 25

Transformation

Question 26

DNA fragments used for molecular cloning come from two sources

Answer 26

Genomic DNA and cDNA both kinds can be manipulated by mutation as well (site-directed mutagenesis)

Question 27

Genomic DNA

Answer 27

from chromatin in the nucleus

Question 28

cDNA

Answer 28

copy DNA or complementary DNA from reverse transcription of mRNA

Question 29

genomic library

Answer 29

a collection of DNA fragments that comprise the genome of an organism ``` The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector, which is used to produce a colony of recombinant cells. ```

Question 30

cDNA libraries

Answer 30

Represent the mRNA population from a given cell/tissue. cDNA is produced by making a DNA copy of the mRNA population using the RNA-directed DNA polymerase called, reverse transcriptase (RTCase). snapshot of the transcription pattern of the cell used to compare gene expression in different tissues at different stages of development to provide the ORF for expressing the protein

Question 31

expression vectors

Answer 31

sequences needed for | expression of a transgene in a host cell.

Question 32

expression vectors for prokaryote host

Answer 32

a bacterial promoter, ribosome binding site, and a transcription termination signal

Question 33

expression vectors for eukaryotic host

Answer 33

eukaryotic promoter/enhancer and terminator (poly-A addition signal/site)

Question 34

other examples of expression vectors

Answer 34

Inducible promoters which respond to a specific signal • Tissue-specific promoters expressed only in certain tissues at certain times • Signal sequences—e.g., a signal to secrete the product to the extracellular medium

Question 35

DNA microarray

Answer 35

provides a large array of | sequences for hybridization experiments.

Question 36

Ti Plasmid

Answer 36

tumor inducing used as a vector for plant cells plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants. ``` rDNA inserted into Ti plasmids, then used to transform Agrobacterium cells, can allow inserted DNA into the plant chromosomes with the T DNA. Whole plants can be regenerated with new functions ```

Question 37

conventional breeding

Answer 37

many generations | only genes from same species can be used

Question 38

biotechnology

Answer 38

one generation; | genes from any organism can be sued