Ch. 3 Recombinant DNA Technology

Question 1

DNA isolation from E. coli

Answer 1

• chemical extraction using phenol removes proteins from the DNA
• RNA is removed from the sample by RNase treatment
• lysozyme digests peptidoglycan in the bacterial cell wall
• centrifugation separates cellular components based on size

Question 2

Agarose gel electrophoresis

Answer 2

Used electric current to separate DNA molecules by size

Question 3

SyBr safe

Answer 3

Sits on the minor grooves of DNA

Question 4

Ethidium bromide

Answer 4

Intercalates between the bases of DNA and RNA, when exposed to UV light

Question 5

DNA isolation and purification

Answer 5

• cell lysis
• extraction of proteins and cellular materials
• phenol extraction and removal of RNA
• DNA precipitation
• centrifugation and washing the DNA pellet

Question 6

___ ___ are naturally occurring enzymes that recognize a particular sequence and cut the phosphate backbone.

Answer 6

Restriction enzymes

Question 7

Cell lysis

Answer 7

• mechanical
• detergent lysing membrane
• DNA will be released in the solutions

Question 8

Extraction of proteins and cellular materials

Answer 8

• centrifugation

- spin column

Question 9

DNA precipitation

Answer 9

Alcohol

Isopropyl alcohol

Question 10

The concentration of DNA or RNA in a liquid can be determined by measuring the..

Answer 10

Absorbance of UV light

Question 11

The absorbance of UV light depends on the..

Answer 11

Amount of DNA and the molecular structure

Question 12

The concentration of DNA or RNA can be determined by..

Answer 12

Measuring the absorbance of UV light at OD260

Question 13

Radioactive labeling is normally used for labeling ___ ___.

Answer 13

DNA probes

Question 14

Radioactively labeled DNA is considered ___, whereas unlabeled DNA is considered ___.

Answer 14

Hot, cold

Question 15

Fluorescent tagged nucleotide was developed as a better method of ___ ___.

Answer 15

DNA detection

Question 16

Atoms were excited and then return to ground state. The released photon is detected with a ___.

Answer 16

Photodetector

Question 17

___ ___ forms hybrid DNA molecules to determine homology using target DNA and probe molecule.

Answer 17

Southern blot

Question 18

___ ___ ___ begins with spotting DNA or RNA samples onto a nylon membrane.

Answer 18

Dot blot hybridization

Question 19

___ is a technique in which a labeled probe is incubated with cells that have had their DNA denatured by heat.

Answer 19

FISH

Question 20

___ ___ are specialized plasmids that will hold and piece of foreign DNA for further study, manipulation, or expression.

Answer 20

Cloning vectors

Question 21

In cells without an insert in the plasmid,

Answer 21

B-gelactosidase is active and splits X-gal to form a blue dye

Question 22

Cells with insert,

Answer 22

B-galactosidase is inactive and cells remain white

Question 23

Cosmid

Answer 23

• behave as plasmid and phage

- nearly all of genome removed so there is room for large inserts

Question 24

Electroporation

Answer 24

• cell suspension in electroporation cuvette

Question 25

Construction of library of genes

Answer 25

• isolate the chromosomal DNA from an organism
• digest the DNA with one or two different restriction enzymes
• linearize a suitable cloning vector with compatible restriction enzyme sites
• mix the cut chromosome fragments with linearized vector and ligate
• transform this mixture into E. coli
• isolate large numbers of E. coli transformants

Question 26

Southern blot

Electrophoresis provides information on:

Answer 26

• size of fragments

- presence of specific sequences

Question 27

___ ___ is added to the nylon membrane where it hybridizes to its matching sequence.

Answer 27

Radioactive probe

Question 28

Identifying a specific clone with a specific probe

Answer 28

• polynucleotides

- antibodies

Question 29

Need to lower ___ of hybridization conditions to tolerate some mismatches.

Answer 29

Stringency

Question 30

High stringency -

Answer 30

Adjust conditions until only perfectly matched DNA strands form a duplex

Question 31

Screening by immunological assay

Answer 31

• the membrane is treated with primary antibody that binds only to the target protein
• unbound primary antibody is washed away, and the membrane is treated with secondary antibody
• unbound secondary antibody is washed away and a colorimetric is carried our to identify the clone

Question 32

Screening by protein activity

Answer 32

From each discrete colony formed on a master plate, a sample is transferred to a solid matrix, such as nitrocellulose or nylon membrane

Question 33

Screening by functional complementation

Answer 33

• defective host cells are transformed with plasmids from genomic library derived from wildtype strain
• the transformed cells that carry a cloned gene that confers the A+ function will grow on minimal medium and selected

Question 34

A ___ ___ is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time

Answer 34

cDNA library

Question 35

Partially degrade the mRNA using ___.

Answer 35

Ribonuclease H

Question 36

Transformation -

Answer 36

Introduce recombinant plasmids into bacterial host cells

Question 37

Select transformants using ___ ___.

Answer 37

Blue-white screening

Question 38

A gene of interest is flanked by sequences homologous to the ___.

Answer 38

BAC